Systems and methods for sample use maximization

ABSTRACT

The present invention provides systems, devices, and methods for point-of-care and/or distributed testing services. The methods and devices of the invention are directed toward automatic detection of analytes in a bodily fluid. The components of the device can be modified to allow for more flexible and robust use with the disclosed methods for a variety of medical, laboratory, and other applications. The systems, devices, and methods of the present invention can allow for effective use of samples by improved sample preparation and analysis.

CROSS-REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/435,250, filed Jan. 21, 2011, which application is entirelyincorporated herein by reference.

BACKGROUND OF THE INVENTION

The discovery of a vast number of disease biomarkers, new therapies andthe establishment of miniaturized medical systems have opened up newavenues for the prediction, diagnosis and monitoring of treatment ofdiseases in a point-of-care or other distributed test settings.Point-of-care systems can rapidly deliver test results to medicalpersonnel, other medical professionals and patients. Early diagnosis ofa disease or disease progression and monitoring of therapy are oftencritical for treatment of deadly conditions such as certain cancers andinfectious diseases.

Diagnosis and treatment of diseases can take advantage of multiplexedbiomarker measurements, which provide additional knowledge of thecondition of a patient. For example, when monitoring the effects of adrug, three or more biomarkers can be measured in parallel. Typically,microtiter plates and other similar apparatuses have been used toperform multiplexed separation-based assays. A microtiter plate (forexample, a 384 well microtiter plate) can perform a large number ofassays in parallel.

In a Point-of-Care (POC) device, the number of assays that can beperformed in parallel is often limited by the size of the device and thevolume of the sample to be analyzed. In many POC devices, the numberassays performed is about 1 to 10. A POC device capable of performingmultiplexed assays on a small sample would be desirable.

A shortcoming of many multiplexed POC assay devices is the high cost ofmanufacturing the components of the device. If the device is disposable,the cost of the components can make the manufacturing of a POC deviceimpractical. Further, for multiplexed POC devices that incorporate allof the necessary reagents onboard of the device, if any one of thosereagents exhibit instability, an entire manufactured lot of devices mayhave to be discarded even if all the other reagents are still usable.

When a customer is interested in customizing a POC device to aparticular set of analytes, manufacturers of multiplexed POC assaysystems are often confronted with the need to mix and match the assaysand reagents of the device. A multiplexed POC assay suitable to eachcustomer can be very expensive, difficult to calibrate, and difficult tomaintain quality control.

POC methods have proven to be very valuable in monitoring disease andtherapy (for example, blood glucose systems in diabetes therapy,Prothrombin Time measurement in anticoagulant therapy using Warfarin).By measuring multiple markers, it is believed that complex diseases(such as cancer) for which multi-drug therapies are required can bebetter monitored and controlled.

There exists the need to use multiple sources of information formonitoring the health status or disease condition of individuals as wellas treatments of various diseases. Especially important is themeasurement of concentrations of several selected analytes (biomarkers,antibodies, gene expression levels, metabolites, therapeutic drugconcentrations and the like) over time. To make this process convenientand maximally effective, technologies that enable measurement of any andall needed analytes (of whatever types) using a small blood sample(blood drop obtained by finger-stick) or other suitable sample areparticularly valuable. Such technology will ideally be operable bynon-technically trained users in distributed test settings, e.g., homes,clinics, doctor's offices, pharmacies, and retail shops. The presentinvention addresses these issues and allows for one to be able to makesuch measurements routinely in patient's home or other non-laboratorysetting.

There also exists the need to make the greatest use of availablesamples, particularly in the instance where samples (e.g., bloodsamples) are limited by sample size. Blood samples are used for thegreat majority of medical/clinical tests. Blood cells have to beseparated from plasma (or serum) prior to most types of analysis sincethe presence of cells would compromise the assay chemistries. Forexample, glucose and cholesterol are often measured by color-formingchemistries which would be interfered with by the presence of formedelements, especially red cells, or hemoglobin (from lysed red cells).

Distributed test systems ideally require a small blood sample obtainedby fingerstick methods. Such samples may be as small as 20 microliters(uL) (one drop) or less. Larger volume samples (say up to 200 uL)usually cannot be taken by fingerstick methods without repeated,inconvenient (“milking”) of fingers. Alternatively venous samples ofseveral milliliters (mL) can be taken but this requires a medicallytrained phlebotomist.

It is usually very difficult to perform more than a single assay usingsmall blood sample with 20 uL or less. This is especially so when theblood sample has to be filtered to remove cells and the recovery ofusable plasma from such small volumes is inefficient. Typically onlyabout 5 uL or less of plasma can be recovered. Samples as large as 200uL can be efficiently separated by automated POC systems (Abaxis,Biosite etc.) but this cannot be done routinely unless a technician isavailable to draw the sample.

SUMMARY OF THE INVENTION

In view of the limitations of current methods, there is a pressing needfor improved methods of automatically separating plasma and/or othermaterials from blood cells. There is also a need for improved accuracyof these measurements on analyte concentration. In measurements ofbiomarkers and other components of blood for the purposes of monitoringtherapy and diagnosis, it is important that the correct volume of samplebe used. In a laboratory setting, this is achieved by utilizing complexautomated instruments and skilled professional staff members. Incontrast in “point-of-care” settings such as homes, retail pharmaciesand shops, and the like, the methods and equipment used must enablenon-technically trained people reliably to obtain and process samples.

The present invention addresses the aforementioned needs and providesrelated advantages.

In some embodiments, the present invention relates to point-of-careand/or point-of-service devices. In some embodiments, the presentinvention relates to systems, devices, user interfaces, and methods forassaying samples using a point-of-care and/or point-of-service device.

In one aspect, the devices and methods disclosed herein are designed toidentify the sample type (blood versus plasma and etc.) to measure thevolume of sample early enough in the assay procedure to ensure anappropriate sample is used is an intended assay. In another aspect, thepresent invention also allows for one to be able to correct forsignificant volume errors that occur in performing an assay.

In yet another aspect, this invention allows for simultaneousmeasurements on several analytes of different types with high accuracy.

An aspect of the invention may be directed to an automated system forseparating one or more components in a biological fluid. The automatedsystem may comprise a pipette tip or closed tube adapted to engage withan aspirator wherein said pipette tip or tube comprises two opposingends, at least one of which is closed or sealable; and a centrifugeconfigured to receive said sealed pipette tip or closed tube to effectsaid separating of one or more components in a biological fluid. In anembodiment, the one or more components are selected from the groupconsisting of blood plasma, blood serum, blood cells, and particulates.In another embodiment, when the pipette tip is engaged with theaspirator to effect a draw of the biological fluid. In anotherembodiment, the pipette tip has an open end that forms an airtight sealwith the aspirator. In another embodiment, the system further comprisesan imaging device; and at least one other pipette tip dimensioned toallow dispensing of a liquid into the pipette tip or tube of (a) or toallow the aspiration of a liquid from the pipette tip or tube of (a). Inanother embodiment, the pipette tip or closed tube is orientedvertically when the centrifuge is at rest. In another embodiment, thepipette tip or closed tube is oriented horizontally when the centrifugeis spinning at a predetermined rotational velocity.

Another aspect of the invention may be a method for isolating componentsin a sample comprising one or more of the following steps: loading asample into a pipette tip or a tube comprising two opposing ends, atleast one of which is sealable or sealed; sealing the pipette tip on theat least one end of the pipette tip; centrifuging the sealed pipettetip, thereby forming an interfacial region that separates the sampleinto a supernatant and a pellet; imaging the centrifuged pipette tip todetermine the location of the interfacial region; and automaticallyaspirating the supernatant based on the location of the interfacialregion. In an embodiment, the method further comprises determining thelocation of the supernatant by said imaging step and automaticallyaspirating the supernatant based on the location of the supernatant. Inanother embodiment, the determination occurs with the aid of aprocessor, and said processor provides instructions to an aspiratingdevice which performs the automated aspiration step. In anotherembodiment, the imaging occurs by use of a camera that is configured tocapture the image of the side profile of the pipette tip or the tube. Inanother embodiment, the supernatant includes one or more of thefollowing: blood plasma or blood serum. In another embodiment, thepellet includes one or more of the following: blood cells orparticulates.

A computer-assisted method for characterizing an analyte suspected to bepresent in a sample may be provided in accordance with an additionalaspect of the invention. The computer-assisted method may compriseobtaining a digital image of the sample, wherein the digital imagecomprises at least a two-dimensional array of pixels, and wherein eachpixel comprises a plurality of intensity values, each of whichcorresponds to a distinct detection spectral region; correlating, withthe aid of a programmable device, the obtained intensity values with apredetermined set of values that define a dynamic range of eachdetection spectral region; and predicting the presence and/or quantityof said analyte in the sample based on said correlating of the obtainedintensity values with a predetermine set of values. In an embodiment,the plurality of intensity values comprise intensity values for red,green, and blue detection spectral regions. In another embodiment, themethod further comprises selecting an illumination wavelength, andilluminating the sample with the selected illumination wavelength priorto and/or concurrently with obtaining the digital image. In anotherembodiment, the method further comprises, subsequent to obtaining thedigital image, (a) selecting another illumination wavelength; (b)illuminating the sample with the other selected illumination wavelength;(c) obtaining another digital image of the sample, wherein the digitalimage comprises at least a two-dimensional array of pixels, and whereineach pixel comprises a plurality of intensity values, each of whichcorresponds to a distinct detection spectral region; and (d) predictingthe presence and/or quantity of said analyte in the sample based on theobtained intensity values from the digital image and said anotherdigital image.

Also, an aspect of the invention may be directed to a method ofmeasuring an analyte concentration in a sample fluid comprisingproviding the sample contained in a container dimensioned with aplurality of distinct widths to permit transmission of light along aplurality of varying path lengths that correspond to the plurality ofdistinct widths; illuminating the container along at least one of theplurality of path lengths; and imaging the container to measure a firstlight intensity transmitted across said at least one of the plurality ofpath lengths, for the determination of the concentration of the analytebased on the measured first light intensity.

In accordance with another aspect of the invention, a method ofdetecting the presence or concentration of an analyte in a sample fluidcontained in a container (e.g., cuvette) may comprise illuminating thecontainer along a first region having a first path length to yield afirst measurement of light intensity transmitted across the first pathlength; moving the sample fluid to another region in the containerhaving another path length if the first measurement falls outside apredetermined dynamic range of transmitted light intensity; illuminatingthe container along the another region to yield another measurement oflight intensity transmitted across the another path length; andoptionally repeating second and third steps until a measurement of lightintensity falls within the predetermined dynamic range, therebydetecting the presence or concentration of the analyte. In anembodiment, the method further comprises deconvoluting a line scan ofthe image, thereby detecting the presence or concentration of ananalyte. In another embodiment, the sample is moved from a first regionof the container having a first path length to a second region of thecontainer having another path length by aspirating the sample. Inanother embodiment, an end of the container is attached to a pipettewhich is configured to aspirate the sample. In another embodiment, thesample is moved up or down the length of the container. In anotherembodiment, the container is a pipette tip. In another embodiment, thecontainer is conically shaped. In another embodiment, the container hastwo open ends. In another embodiment, a first open end has a greaterdiameter than a second open end. In another embodiment, the containerhas a plurality of distinct widths to permit transmission of light alonga plurality of varying path lengths. In another embodiment, thecontainer volume is less than 100 microliters. In another embodiment, aplurality of distinct path lengths are imaged simultaneously.

A method may be provided as an additional aspect of the invention. Themethod may be provided for characterizing an analyte suspected to bepresent in a sample of biological fluid, said method comprising:providing said sample of biological fluid; allowing said analyte toreact with one or more reagents that specifically react with saidanalyte to generate an optically detectable signal; and measuring saidoptically detectable signal with a plurality of detection spectralregions, wherein the presence of said optically detectable signal withina dynamic range of at least one detection spectral region is indicativeof the concentration of said analyte in said sample of biological fluid.In an embodiment, the measuring is performed by an imaging deviceconfigured to measure a plurality of detection spectral regions. Inanother embodiment, the imaging device is configured to measure theplurality of detection spectral regions simultaneously. In anotherembodiment, the imaging device is configured to measure the plurality ofdetection spectral regions sequentially.

An aspect of the invention provides a method for increasing the accuracyof an assay comprising imaging a sample in a first tip to determine thevolume of the first sample; imaging one or more reagents in a second tipto determine the volume of the one or more reagents; mixing the sampleand the one or more reagents to form a reaction mixture; imaging thereaction mixture; correcting a calibration based on said determinedvolumes of the sample and the one or more reagents; and calculating aconcentration of an analyte using the corrected calibration. In anembodiment, the method further comprises imaging the reaction mixture todetermine the volume of the reaction mixture. In another embodiment, theimaging of the sample in the first tip is conducted using a cameraconfigured to capture a side profile of the first tip. In anotherembodiment, imaging of the one or more reagents in the second tip isconducted using a camera configured to capture a side profile of thesecond tip. In another embodiment, the height of the sample and the oneor more reagents is calculated based on the captured profiles. Inanother embodiment, determining the volume is based on the height of thesample and the one or more reagents and the known cross-sectional areasof the sample and the one or more reagents respectively. In anotherembodiment, the calibration is also based on the determined volume ofthe reaction mixture.

Another aspect of the invention provides a setup, comprising: a vesselconfigured to accept and confine a sample, wherein the vessel comprisesan interior surface, an exterior surface, an open end, and an opposingclosed end; and a tip configured to extend into the vessel through theopen end, wherein the tip comprises a first open end and second openend, wherein the second open end is inserted into the vessel, whereinthe vessel or the tip further comprises a protruding surface featurethat prevents the second open end of the tip from contacting the bottomof the interior surface of the closed end of the vessel. In anembodiment, the surface feature is integrally formed on the bottominterior surface of the vessel. In another embodiment, the surfacefeature comprises a plurality of bumps on the bottom interior surface ofthe vessel. In another embodiment, the protruding surface feature is ator near the closed end.

Another aspect of the invention provides a sample processing apparatuscomprising a sample preparation station, assay station, and/or detectionstation; a control unit having computer-executable commands forperforming a point-of-service service at a designated location with theaid of at least one of said sample preparation station, assay stationand detection station; and at least one centrifuge configured to performcentrifugation of a sample from a fingerstick. In an embodiment, thecentrifuge is contained within the sample preparation station and/or theassay station. In another embodiment, the computer-executable commandsare configured to perform the point-of-service service at a siteselected from the group consisting of a retailer site, the subject'shome, or a health assessment/treatment location.

Another aspect of the invention provides a method for dynamic feedback,said method comprising: taking an initial measurement of a sample withina container using a detection mechanism; based on said initialmeasurement, determining, using a processor, whether the sampleconcentration falls into a desired range, and determining, using aprocessor, (a) a degree of dilution to be performed if the sampleconcentration is higher than the desired range or (b) a degree ofconcentration to be performed if the sample concentration is lower thanthe desired range; and adjusting the sample concentration according tothe determined degree of dilution or the determined degree ofconcentration. In an embodiment, the method further comprises taking asubsequent measurement of the sample within the container. In anotherembodiment, the method further comprises, based on the subsequentmeasurement determining, using a processor, whether the sampleconcentration falls into a desired range. In another embodiment, thesubsequent measurement is made using the detection mechanism. In anotherembodiment, the method further comprises determining a characteristic ofthe sample based on the subsequent measurement. In another embodiment,the characteristic is selected from one or more of the following: thepresence or concentration of an analyte, the presence or concentrationof a cell, and the morphology of the cell. In another embodiment, thesubsequent measurement is made using a separate detection mechanism fromthe initial detection mechanism. In another embodiment, the initialmeasurement provides a crude cell concentration measurement of thesample. In another embodiment, the subsequent measurement provides ameasurement of cell concentration of the sample of greater resolutionthan the initial measurement. In another embodiment, the initialmeasurement is taken by imaging the sample. In another embodiment, theadjusting of the sample concentration permits detection of analyte thatwould otherwise fall outside the desired range.

Another aspect of the invention provides a method for providing qualitycontrol, said method comprising capturing an image of conditions underwhich a detection mechanism measures a characteristic of a sample; anddetermining, using a processor, based on the image whether there areundesirable conditions under which the detection mechanism is operated.In an embodiment, the undesirable conditions includes the presence ofone or more undesirable materials. In another embodiment, theundesirable materials includes one or more of the following: bubbles,particles, fibers, debris, and precipitates that interfere with themeasurement of the characteristic of the sample. In another embodiment,the detection mechanism is a different mechanism from a mechanism usedto capture the image. In another embodiment, the image is captured usinga camera. In another embodiment, the method further comprises providingan alert if an undesirable condition is detected. In another embodiment,the method further comprises adjusting the sample if an undesirablecondition is detected. In another embodiment, the image includes animage of the sample. In another embodiment, the image includes an imageof one or more of the following: the sample container or the detectionmechanism.

Another aspect of the invention is an automated system for separatingone or more components in a biological fluid comprising a centrifugecomprising one or more bucket configured to receive a container toeffect said separating of one or more components in a fluid sample; andthe container, wherein the container includes one or more shaped featurethat is complementary to a shaped feature of the bucket. In anembodiment, the shaped feature of the bucket includes one or more shelfupon which a protruding portion of the container is configured to rest.In another embodiment, the bucket is configured to be capable ofaccepting a plurality of containers having different configurations, andwherein the shaped feature of the bucket includes a plurality ofshelves, wherein a first container having a first configuration isconfigured to rest upon a first shelf, and a second container having asecond configuration is configured to rest upon a second shelf.

Another aspect of the invention provides an assay unit comprising afirst end and a second end; an outer surface; and an inner surfacecomprising one or more selected patterns each of which is immobilizedthereon or therein with a capture reagent capable of capturing ananalyte suspected to be present in a biological sample, wherein thefirst end and the second end are of different dimensions.

Another aspect of the invention provides an assay unit comprising anidentifier that is used to determine (a) the one or more capturereagents immobilized on the inner surface; and (b) source of thebiological sample if the assay unit contains said sample.

Another aspect of the invention provides an assay unit comprising aplurality of selected patterns, each pattern of said plurality comprisesa distinct capturing agent.

Other goals and advantages of the invention will be further appreciatedand understood when considered in conjunction with the followingdescription and accompanying drawings. While the following descriptionmay contain specific details describing particular embodiments of theinvention, this should not be construed as limitations to the scope ofthe invention but rather as an exemplification of preferableembodiments. For each aspect of the invention, many variations arepossible as suggested herein that are known to those of ordinary skillin the art. A variety of changes and modifications can be made withinthe scope of the invention without departing from the spirit thereof.The various compounds/devices disclosed herein can be used separately orconjunctively in any combination, for any methods disclosed herein aloneor in any combinations.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawing(s) of which:

FIG. 1 shows a side view of a centrifuge.

FIG. 2 shows a face on view of a centrifuge.

FIG. 3 shows a perspective view of the back of a centrifuge.

FIG. 4 shows a top view of a sample tip.

FIG. 5 shows a side view of a sample tip.

FIG. 6 shows a cross-sectional view of a sample tip.

FIG. 7 shows a diagram of a sample tip positioned in a sample above aplasma/packed cell interface.

FIG. 8 shows a graph of centrifugation time as a function of revolutionsper minute.

FIG. 9 shows a graph of centrifugation time as a function of the radiusof the centrifuge rotor.

FIG. 10 shows an empty capped sample tip.

FIG. 11 shows a capped sample tip containing a sample of a bodily fluid,e.g., blood.

FIG. 12 shows a capped sample tip containing a sample of about 23%hematocrit blood after centrifugation.

FIG. 13 shows a capped sample tip containing a sample of about 31%hematocrit blood after centrifugation.

FIG. 14 shows a capped sample tip containing a sample of about 40%hematocrit blood after centrifugation.

FIG. 15 shows a capped sample tip containing a sample of about 52%hematocrit blood after centrifugation.

FIG. 16 shows a capped sample tip containing a sample of 68% hematocritblood after centrifugation.

FIG. 17 shows a comparison of hematocrit measured using by digitallyimaging system a centrifuged sample (“hematocrit, % reported”) andhematocrit measured by standard microhematocrit apparatus (“hematocrit,% target”)

FIG. 18 shows a diagram of a tip used for reactions and a tip used forblood/plasma (dimensions shown in mm).

FIG. 19 shows a cylindrical capillary containing a sample.

FIG. 20 shows angles and dimensions for calculating volumes within aconical container, e.g. a capillary.

FIG. 21 shows angles and dimensions for calculating volumes within aconical container, e.g., a capillary.

FIG. 22 shows angles and dimensions for calculating volume of aspherical cap.

FIG. 23 shows dimensions for calculating the volume of a samplecontained within a cylindrical tip, where the sample has a singlemeniscus.

FIG. 24 shows dimensions for calculating the volume of a samplecontained within a cylindrical tip, where the sample has two menisci.

FIG. 25 shows dimensions for calculating the volume of a samplecontained within and/or associated with a cylindrical tip, where thesample has two menisci and one of which is external to the cylindricaltip.

FIG. 26 shows dimensions for calculating the volume of a samplecontained within a cylindrical tip, where there is a bubble in thesample.

FIG. 27 shows dimensions for calculating the volume of a samplecontained within a cylindrical tip, where there is a bubble in thesample that spans the width of the cylindrical tip.

FIG. 28 shows dimensions for calculating the volume of a samplecontained within and/or associated with a cylindrical tip, where thesample includes a pendant droplet of sample outside the cylindrical tip.

FIG. 29 shows dimensions for calculating the volume of a residual samplecontained within a cylindrical tip.

FIG. 30 shows a blood sample within a tip prior to being mixed with amagnetic reagent.

FIG. 31 shows a blood sample being mixed with a magnetic reagent.

FIG. 32 shows a blood sample mixed with a magnetic reagent.

FIG. 33 shows a blood sample mixed with a magnetic reagent containedwithin a tip.

FIG. 34 shows a blood sample mixed with a magnetic reagent moved to aselected position within a tip.

FIG. 35 shows a magnetic force applied by a magnet (M) to a blood samplemixed with a magnetic reagent.

FIG. 36 shows a blood sample that has been separated into a red cellcomponent and a plasma component using magnetic force.

FIG. 37 shows a well positioned beneath a tip containing a blood samplethat has been separated into a red cell component and a plasmacomponent.

FIG. 38 shows a depiction of blood plasma being transferred from a tipto a well.

FIG. 39 shows a tip after dispensing of blood plasma to a well.

FIG. 40 shows a high contrast image of a cylindrical tip containing aliquid with low absorbance.

FIG. 41 shows an image of a conical tip containing a liquid with highabsorbance.

FIG. 42 shows a tip with a high absorbance liquid showing two menisciwithin the tip.

FIG. 43 shows a tip with a sample liquid and large bubbles that span thediameter of the tip.

FIG. 44 shows a tip containing water showing a clear upper meniscus in atransparent tip or capillary.

FIG. 45 shows a graph of computed Protein-C concentration as a functionof sample volume.

FIG. 46 shows an image of a sample transfer device with a capillary,housing, plunger, groove, and raised feature. The raised feature mayhelp locate the plunger in the housing.

FIG. 47 shows a sample contained with the capillary of a sample transferdevice.

FIG. 48 shows a sample transfer device after a sample has been ejectedby a plunger.

FIG. 49 shows a sample transfer device after a sample has beenincompletely ejected.

FIG. 50 shows a conical tip containing a sample, with the position L3indicated by the arrow shown.

FIG. 51 shows a graph of the ratio of the distance between L2 and L1 andthe distance between L3 and L1 as a function of sample volume.

FIG. 52 shows a schematic of a chemical reaction that produces a coloredproduct.

FIG. 53 shows a schematic of a chemical reaction that produces a coloredproduct from cholesterol.

FIG. 54 shows a schematic of a chemical reaction that uses reducingequivalents to produce a colored product.

FIG. 55 shows an example of a compound that changes color upon beingcomplexed with a metal ion.

FIG. 56 shows a series of images of tips with two-fold decreasingconcentration of albumin from right to left, except for the left-mosttip, which has no albumin.

FIG. 57 shows a series of images of tips with two-fold decreasingconcentration of cholesterol from right to left, except for theleft-most tip, which has no cholesterol.

FIG. 58 shows a series of hemispherical wells machined from a block ofwhite opaque plastic, which each well having two-fold decreasingconcentration of analyte from right to left, except for the left-mostwell, which has no analyte. In some embodiments, the analyte may becalcium.

FIG. 59 shows a series of hemispherical wells machined from a block ofwhite opaque plastic, which each well having two-fold decreasingconcentration of analyte from right to left, except for the left-mostwell, which has no analyte. In some embodiments, the analyte may bemagnesium.

FIG. 60 shows a series of hemispherical wells machined from a block ofwhite opaque plastic, which each well having two-fold decreasingconcentration of analyte from right to left, except for the left-mostwell, which has no analyte. In some embodiments, the analyte may beurea.

FIG. 61 shows a series of tips containing bromophenol blue solutions.

FIG. 62 is an illustration of tips having a plurality of distinctoptical path lengths.

FIG. 63 shows a light path through a rectangular cuvette.

FIG. 64 shows a light path through a microtiter well.

FIG. 65 shows a light path through a conically shaped cuvette.

FIG. 66 shows a graph of light intensity as a function of location asmeasured on tips containing samples with varying concentration ofbromophenol blue solutions for red, green, and blue color channels.

FIG. 67 shows an image of the tips that were analyzed in FIG. 66.

FIG. 68 shows a graph of signal as a function of bromophenol blueconcentration as measured by red, green, and blue color channels. Theoptical density may be measured at 589 nm.

FIG. 69 shows a log scale graph of signal response as a function ofbromophenol blue concentration as measured by blue (diamonds) and red(squares) color channels.

FIG. 70 shows a graph of concentration measured by color analysis ofdigital images as a function actual concentration.

FIG. 71 shows a graph of signal response as measured by red (squares),green (diamonds), and blue (triangles) color channels as a function ofalbumin concentration.

FIG. 72 shows three graphs of signal response as measured for green,red, and blue color channels for polystyrene latex particles.

FIG. 73 shows tips that each separately contain reagents NADH, WST-1,PMS, and two tips containing a mixture of the reagents.

FIG. 74 shows a digital image of tips containing two-fold decreasingconcentration of lactate dehydrogenase (LDH) from left to right.

FIG. 75 shows a graph of optical density measured at 450 nm as afunction of LDH.

FIG. 76 shows solutions of potassium chloride added to potassium assaystrips.

FIG. 77 shows tips containing blood samples mixed with blood typingreagents for Anti-A, Anti-B, Anti-D, and Control (from left to right).

FIG. 78 shows measured signals for signal as a function of position forred (left column), green (middle column), and blue (right column) forsamples mixed with Anti-A, Anti B, Anti-D, and Control reagents.

FIG. 79 shows normalized signal as a function of relative concentrationmeasured for narrow and wide path lengths using red, green, and bluecolor channels.

FIG. 80 shows a graph of log of measured concentration as a function ofactual concentration, illustrating the accuracy of the measurementalgorithm.

FIG. 81 shows a fluorescence image of assay products in tubes.

FIG. 82 shows an image of reaction products in tips.

FIG. 83 shows an image of reaction products in tips.

FIG. 84 shows an image of reaction products in tips.

FIG. 85 shows an image of reaction products in tips.

FIG. 86 shows an image of reaction products in tips.

FIG. 87 shows an image of reaction products in tips.

FIG. 88 shows a background color image obtained for calibration.

FIG. 89 shows a fluorescence image of reaction products in tips.

FIG. 90 shows red and blue color channel response and fluorescenceresponse as a function of DNA copy number.

FIG. 91 shows graph of transformed 3-color signal as a function offluorescence signal.

FIG. 92 shows a graph of green channel signal response as a function ofpixel position.

FIG. 93 shows an image of tips containing solutions of bromophenol blueand water.

FIG. 94 shows an image of additional tips that may contain solutions ofbromophenol blue and water.

FIG. 95 shows an schematic of a tip containing reaction mixtures toperform multiple assays.

FIG. 96 shows an image of tips containing solutions of bromophenol blueand water.

FIG. 97 shows a graph of signal response for sample, water, and controlin multiple standards. The samples may be aqueous calibrators containingknown concentrations of analyte.

FIG. 98 shows tips containing assays for both Ca²⁺ (upper region of thetip) and Mg²⁺ (lower region of the tip).

FIG. 99 shows four tips with various types of serum samples: hemolyzed(reddish in color), lipemic (gray), icteric (yellow in color), andnormal (from left to right).

FIG. 100 shows a schematic of a camera and optical components.

FIG. 101 shows a cross-sectional view of a camera and optical componentsincluding a white light source, an aperture, and a sensor.

FIG. 102 shows a schematic of an optical setup for measuring lightsignal using (A) a sensor that is positioned to detect light at aperpendicular angle to an excitation beam, and (B) a sensor that ispositioned in line with an excitation beam.

FIG. 103 shows images taken using (A) an excitation beam perpendicularto a sensor and (B) an excitation beam that is in line with a sensor.

FIG. 104 shows an array of printed dyes that can be used to calibratethe optical setup.

FIG. 105 shows a graph of signal as a function of sample volume. Series1-5 may correspond to different analyte concentrations, such as 0, 20,40, 60, and 80 respectively.

FIG. 106 shows a graph of signal as a function of sample volume. Series1-5 may correspond to different analyte concentrations, such as 0, 20,40, 60, and 80 respectively.

FIG. 107 shows a graph of signal as a function of sample volume. Series1-5 may correspond to different analyte concentrations, such as 0, 20,40, 60, and 80 respectively.

FIG. 108 shows a graph of measured analyte concentration as a functionof actual analyte concentration.

FIG. 109 shows a graph of measured analyte concentration as a functionof actual analyte concentration.

FIG. 110 schematically illustrates an exemplary method for an ELISAassay.

FIG. 111 shows an example of a rotor at rest with buckets vertical.

FIG. 112 shows an example of a rotor at a speed with buckets at a smallangle to horizontal.

FIG. 113 shows an example of a bucket configuration.

FIG. 114 shows an example of a centrifugation vessel mated with thebucket.

FIG. 115 shows an example of another centrifugation vessel that can bemated with the bucket.

FIG. 116 shows an example of a centrifugation vessel.

FIG. 117 shows an example of an extraction tip.

FIG. 118 provides an example of how the centrifugation vessel andextraction tip may mate.

FIG. 119 is an image that was taken of the original reaction mixtureprior to centrifugation.

FIG. 120 is another image that was taken of the original reactionmixture prior to centrifugation

FIG. 121 is an additional image that was taken of the original reactionmixture prior to centrifugation

FIG. 122 shows results as distance of the interface from the plasmameniscus.

FIG. 123 provides an example of a fluorescence micrograph showinglabeled leukocytes.

FIG. 124 provides an example of intracellular patterns using darkfieldimages.

FIG. 125 provides an example of multi-parameter acquisition of data fromlabeled cell samples.

FIG. 126 provides an example of brightfield images of human whole blood.

FIG. 127 provides an example of quantitative multi-parametric dataacquisition and analysis.

FIG. 128 shows variation in light distribution.

FIG. 129 shows data from five assays.

FIG. 130 shows a parameter plotted against concentration of the analyte,as well as graphs relating to accuracy, precision, and predictedconcentration.

FIG. 131 shows images collected by a digital camera.

FIG. 132 illustrates examples of images taken of reaction product.

FIG. 133 provides examples of images that were analyzed before spinningin the centrifuge, and after spinning in the centrifuge.

FIG. 134 illustrates examples of images taken of reaction product.

FIG. 135 illustrates spectra of several serum samples.

FIG. 136 illustrates an example detection process of the invention usingan array.

FIG. 137 illustrates an example detection process of the invention usingbeads.

FIG. 138 illustrates an example detection process of the invention usingtagged aptamers.

FIG. 139 illustrates detection of aptamer binding to a complementaryprobe.

FIG. 140 illustrates absence of binding between aptamer and anon-complementary probe.

FIG. 141 illustrates binding specificity of aptamers on an array.

FIG. 142 shows a more detailed view of analyte detection on an array.

FIG. 143 shows an example array.

FIG. 144 shows a plot of chemiluminescence against concentration for avitamin D assay.

FIG. 145 shows a plot of chemiluminescence against concentration for anestradiol assay.

FIG. 146 shows a spectrophotometric measurement of WBC concentration.

FIG. 147 shows plots of turbidity as a function of time.

FIG. 148 is a plot of inflection points for three experiments at 800copies/uL and 80 copies/uL.

FIG. 149 is a plot of an example in which magnetic beads are used forthe analysis of proteins and small molecules via ELISA assays.

FIG. 150 is a plot of an example in which magnetic beads are used forthe analysis of proteins and small molecules via ELISA assays.

DETAILED DESCRIPTION OF THE INVENTION

While preferable embodiments of the invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein can be employed in practicing the invention.

The invention provides mobile applications for system and methods forsample use maximization. Various aspects of the invention describedherein may be applied to any of the particular applications set forthbelow or for any other types of diagnostic or therapeutic applications.The invention may be applied as a standalone system or method, or aspart of an integrated pre-clinical, clinical, laboratory or medicalapplication. It shall be understood that different aspects of theinvention can be appreciated individually, collectively, or incombination with each other.

The devices and systems herein can provide an effective means forreal-time detection of analytes present in a bodily fluid from asubject. The detection methods may be used in a wide variety ofcircumstances including identification and quantification of analytesthat are associated with specific biological processes, physiologicalconditions, disorders or stages of disorders. As such, the systems havea broad spectrum of utility in, for example, drug screening, diseasediagnosis, phylogenetic classification, parental and forensicidentification, disease onset and recurrence, individual response totreatment versus population bases, and/or monitoring of therapy. Thesubject devices and systems are also particularly useful for advancingpreclinical and clinical stage of development of therapeutics, improvingpatient compliance, monitoring ADRs associated with a prescribed drug,developing individualized medicine, outsourcing blood testing from thecentral laboratory to the home or on a prescription basis, and/ormonitoring therapeutic agents following regulatory approval. The subjectdevices and system can be utilized by payors outsourcing blood testsfrom a central laboratory. The devices and systems can provide aflexible system for personalized medicine. Using the same system, adevice can be changed or interchanged along with a protocol orinstructions to a programmable processor of the systems to perform awide variety of assays as described. The systems and devices describedherein, while being much smaller and/or portable, embody novel featuresand offer many functions of a laboratory instrument.

In an aspect, a system of the invention comprises a device comprisingassay units and reagent units, which include reagents, e.g., both liquidand/or solid phase reagents. In some embodiments, at least one of thewhole device, an assay unit, a reagent unit, or a combination thereof isdisposable. In a system of the invention, the detection of an analytewith the subject device is typically automated. Such automation can beeffected by a built-in protocol or a protocol provided to the system bythe manufacturer.

The devices and systems as described herein can offer many features thatare not available in existing POC systems or integrated analysissystems. For example, many POC cartridges rely on a closed fluidicsystem or loop to handle small volumes of liquid in an efficient manner.The fluidic devices such as cartridges described herein can have openfluid movement between units within a given cartridge. For example, areagent can be stored in a unit, a sample stored in a sample collectionunit, a diluent stored in a diluent unit, and the capture surface can bein an assay unit, where in one state of cartridge, none of the units arein fluid communication with any of the other units. The units can bemovable relative to each other in order to bring some units into fluidcommunication using a fluid transfer device of the system. For example,a fluid transfer device can comprise a head that engages an assay unitand brings the assay unit in fluidic communication with a reagent unit.In some cases, the head is a pipette head that moves the assay unit(e.g., tip) in fluid communication with a reagent unit.

Accordingly, in an embodiment, the present invention provides a methodof detecting and/or measuring the concentration of an analyte in abodily fluid or tissue sample, the method typically comprises the stepsof providing a sample (e.g., blood, urine, saliva, tissue) to a deviceor system of the invention, allowing the sample to react within at leastone assay unit of the device, and detecting the detectable signalgenerated from the analyte in the blood sample.

One aspect of the invention provides for analyzing samples using apoint-of-care device that is configured to maximize sample utilization.For example, more than about 15, 25, 50, 75, or 100 assays can beperformed on a sample having a volume of less than about 1, 20, 50, 100,or 500 μL. The sample can be a blood sample taken from a finger prick.The sample can be collected in a sealable capillary or tip. The samplecan be prepared for one or more assays by subjecting the sample to aseparation (e.g., centrifugation) and/or dilution process. The one ormore assays can be prepared by combining the sample, which may have beenseparated and diluted, with one or more reagents in a reaction chamber.The reaction chamber can be a pipette tip, vial, a sample transferdevice, and/or a cuvette. The one or more assays can be configured suchthat an optical signal can be measured which is indicative of theconcentration of one or more analytes in the sample. The reactionchamber can contain a plurality of assays, which may be spatiallyseparated. A plurality of optical signals can be generated within asingle reaction chamber from one assay, or from a plurality of spatiallyseparated assays. The one or more optical signals can be measured by adigital imaging camera that can measure a plurality of detectionspectral regions or detection bands, e.g., red, green and blue. Theoptical signal can be measured on the assay reaction product in thereaction chamber, which can be a pipette tip or other sample containers.The systems, devices, and methods can be fully automated orsemi-automated by programmable logic.

Another aspect of the invention provides for systems, devices, andmethods for preparing samples for analysis. Samples can be prepared foranalysis by one or more separation devices. For example, a sample can beprepared for analysis by centrifugation within a centrifuge. Otherseparations based on charge, size, hydrophobicity/hydrophilicity, and/orvolatility can also be implemented.

One aspect of the invention provides for sample and reaction productanalysis using image-based analysis. The system can include a camerathat can measure an optical signal using one or more detection spectrumregions. For example, a camera can measure an optical signal using red,green, and blue detection spectrum regions. The measured signal caninclude three measured values that can be interpreted using one or morealgorithms described herein. The use of more than one detection spectrumregion can increase the dynamic range of an assay and can increase theaccuracy of a measurement as compared to measurements using a singledetection spectrum region.

The invention also provides for systems, devices, and methods forperforming optical measurements on samples and assay reaction productsthat are contained within reaction chambers, each with a plurality ofdistinct path lengths. The reaction chambers can have a plurality ofdistinct path lengths such that a greater or lower amount of lightabsorbance is observed. The plurality of distinct path lengths (such as,for example, through the sample and/or reaction chamber) allows for anincrease in the dynamic range of a selected assay protocol. The image ofthe reaction chamber can be analyzed as described herein to obtaininformation on the sample or the assay reaction products. Thecombination of utilizing the plurality of available path lengths withina single reaction chamber and the use of three channel detectionspectrum regions greatly enhances the dynamic range of a given assay.

A system for performing sample preparation and analysis can includeinstrumentation, disposable components, and reagents. The system canaccept samples and automatically performs a plurality of assays withoutuser intervention. Where desired, the instrumentation can include agraphical user interface, a mechanism for introducing cartridges, whichmay be disposable, a motorized stage, which may have mobility in threedimensions, one or more single-head liquid handling devices, one or moremulti-head liquid handling devices, one or more devices for performingsample preparation, optical sensors, which can include a PMT and/or animaging device, temperature controllers, and communication devices. Thedisposable component can include a disposable cartridge that containssample tips, tip seals, and reagents. In some embodiments, thedisposable cartridge may also contain neutralizing assemblies configuredto absorb and neutralize liquid assay products.

The instrumentation, disposable components, and reagents can be housedwithin a closeable environment, such as a case or a cabinet. In someembodiments, the case has a cross-sectional area less than about 4 m², 2m², 1 m², 0.5 m², 0.1 m², 0.05 m², or lower. The invention provides fora distributed test system, such as a point-of-care device, which caninclude one or more of the following aspects:

1. Efficient (centrifugal) separation of blood and recovery of theseparated plasma

2. Dilution of the plasma sample to one or more levels (for example1:10, 1:100, 1:1000) so that each assay can be performed at an optimaldilution

3. Optimized distribution of sample to several different assays whichmay involve several different methodologies

4. Optimal assay protocols

5. Use of open-ended circular section cuvettes for sample analysis,mixing with reagents, incubation and presentation to optical systems

6. Analysis of assays using imaging technology (scanning and/orphotography, and/or microscopy)

In one embodiment, the device of the invention is self-contained andcomprises all reagents, liquid- and solid-phase reagents, required toperform a plurality of assays in parallel. Where desired, the device isconfigured to perform at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 100, 200, 500, 1000 or more assays. One or more control assays canalso be incorporated into the device to be performed in parallel ifdesired. Calibrators can also be provided for assay system calibration.Some examples of dried controls and calibrators useful for assay systemcalibration can include aqueous solutions of analytes, serum, or plasmasamples with known levels of analytes, known quantities of suchcalibrators and controls can also be dried by lyophilization, vacuumdrying, and other manufacturing processes (and dissolved during theassay).

By incorporating these components within a point-of-care system, apatient or user can have a plurality of analytes, for example more thanabout 10, 20, 30, 50, 75, 100, 150, or 200 analytes, quantified withinless than about 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 60, 120, 180, 240, 480or 600 minutes.

The subject devices and systems can be utilized for conductingquantitative immunoassays, which can be completed in a short period oftime. Other assay type can be performed with a device of the inventionincluding, but not limited to, measurements of nucleic acid sequencesand measurements of metabolite, such as cholesterol or electrolytes suchas magnesium and chloride ions. In some embodiments, the assay iscompleted in no more than one hour, preferably less than 120, 60, 30,15, 10, 5, 4, 3, 2, or 1 minute. In other embodiments, the assay isperformed in less than 5 minutes. The duration of assay detection can beadjusted accordingly to the type of assay that is to be carried out witha device of the invention. For example, if needed for highersensitivity, an assay can be incubated for more than one hour or up tomore than one day. In some examples, assays that require a long durationmay be more practical in other POC applications, such as home use, thanin a clinical POC setting.

In other embodiments of the invention the reagent units of a subjectdevice are configured to be a set of mix-and-match components. A reagentunit typically stores liquid or solid reagents necessary for conductingan assay that detect a given analyte. The assay units can sometimes (oroptionally not always) comprise at least one capture surface capable ofreacting with an analyte from the sample of bodily fluid. The assay unitmay be a tubular tip with a capture surface within the tip. Examples oftips of the invention are described herein. Each individual assay andreagent unit can be configured for assay function independently. Toassemble a device, the units can be assembled in a just-in-time fashionfor use in an integrated device, which can take the format of cartridge.

A housing for a device of the invention can be made of a polymericmaterial, a metallic material or a composite material, such as, e.g.,aluminum, polystyrene or other moldable or machinable plastic, and canhave defined locations to place assay units and reagent units. Thehousing may include a metal or any other material. The housing maypartially or entirely enclose the assay units and/or reagent units. Thehousing may support the weight of the assay units and/or reagent units.In an embodiment, the housing has means for blotting tips or assay unitsto remove excess liquid. The means for blotting can be a porousmembrane, such as cellulose acetate, or a piece bibulous material suchas filter paper.

In some embodiments, at least one of the components of the device may beconstructed of polymeric materials. Non-limiting examples of polymericmaterials include polystyrene, polycarbonate, polypropylene,polydimethysiloxanes (PDMS), polyurethane, polyvinylchloride (PVC),polysulfone, polymethylmethacrylate (PMMA),acrylonitrile-butadiene-styrene (ABS), and glass.

The device or the subcomponents of the device may be manufactured byvariety of methods including, without limitation, stamping, injectionmolding, embossing, casting, blow molding, machining, welding,ultrasonic welding, and thermal bonding. In an embodiment, a device inmanufactured by injection molding, thermal bonding, and ultrasonicwelding. The subcomponents of the device can be affixed to each other bythermal bonding, ultrasonic welding, friction fitting (press fitting),adhesives or, in the case of certain substrates, for example, glass, orsemi-rigid and non-rigid polymeric substrates, a natural adhesionbetween the two components.

A system as described can run a variety of assays, regardless of theanalyte being detected from a bodily fluid sample. A protocol dependenton the identity of the device may be transferred from an external devicewhere it can be stored to a reader assembly to enable the readerassembly to carry out the specific protocol on the device. In someembodiments, the device has an identifier (ID) that is detected or readby an identifier detector described herein. The identifier can enabletwo-way communication between the device and a sensor or receivingsystem. The identifier detector can communicate with a communicationassembly via a controller which transmits the identifier to an externaldevice. Where desired, the external device sends a protocol stored onthe external device to the communication assembly based on theidentifier. The protocol to be run on the system may compriseinstructions to the controller of the system to perform the protocol,including but not limited to a particular assay to be run and adetection method to be performed. Once the assay is performed by thesystem, a signal indicative of an analyte in the bodily fluid sample isgenerated and detected by a detection assembly of the system. Thedetected signal may then be communicated to the communications assembly,where it can be transmitted to the external device for processing,including without limitation, calculation of the analyte concentrationin the sample.

Systems, devices and methods for performing sample analysis usingpoint-of-care devices and tips that can function as reaction chambersare described in U.S. Patent Publication No. 2009/0088336 and U.S.Provisional Application No. 60/997,460, each of which is incorporatedherein by reference in its entirety for all purposes.

Sample Handling and Reaction Chambers

Samples, reagents, and assembled assays described herein can be handledand contained by a variety of reaction chamber types. A sample handingdevice and a reaction chamber can be a well, a tube, or an open endedtip, which may also be a cuvette. As used herein, a tip can alsoreferred to as a sample tip, a cuvette tip, a reaction chamber, acuvette, a capillary, a sample handing device, or a sample transferdevice. Samples may be collected from a source into a tip or a tube. Thetips may be sealed. Such seals may be permanent or reversible. Dilutedsamples can be combined with one or more reagents and mixed (asdescribed in previous applications) within “assay elements” such as tips(open-ended cuvettes) or open or covered wells. Once the assay is readyfor reading, the assay element can be presented to the optical systemfor image analysis or other types of reading. Alternatively, assayreaction mixtures can be transferred from one type of element toanother. For example, assays incubated in tips can be blotted onto anabsorbent or bibulous medium or assays incubated in wells can beaspirated into tips. Many assays can be processed in parallel. Assayreadout can be serial or simultaneous depending on the assay protocoland/or incubation time. For assays involving measurement of a rate ofchange, the assay element can be presented to the optical system morethan once at different times.

Fluid and Material Transfer Devices

A fluid transfer device can be part of a system. The fluid transferdevice can comprise a plurality of heads. Any number of heads as isnecessary to detect a plurality of analytes in a sample is envisionedfor a fluid transfer device of the invention. In an example, a fluidtransfer device has about eight heads mounted in a line and separated bya distance. In an embodiment, the heads have a tapered nozzle thatengages by press fitting with a variety of tips, such as assay unit orsample collection units as described herein. The tips can have a featurethat enables them to be removed automatically by the instrument anddisposed into in a housing of a device as described after use. In anembodiment, the assay tips are clear and transparent and can be similarto a cuvette within which an assay is run that can be detected by anoptical detector such as a photomultiplier tube or camera sensor.

In an example, a programmable processor (e.g., central processing unit,CPU) of a system can comprise or be configured to accept (such as, e.g.,from a memory location) instructions or commands and can operate a fluidtransfer device according to the instructions to transfer liquid samplesby either withdrawing (for drawing liquid in) or extending (forexpelling liquid) a piston into a closed air space. The processor can beconfigured to facilitate aspiration and/or dispensing. Both the volumeof air moved and the speed of movement can be precisely controlled, forexample, by the programmable processor.

Mixing of samples (or reagents) with diluents (or other reagents) can beachieved by aspirating components to be mixed into a common tube andthen repeatedly aspirating a significant fraction of the combined liquidvolume up and down into a tip. Dissolution of reagents dried into a tubecan be done is similar fashion. Incubation of liquid samples andreagents with a capture surface on which is bound a capture reagent (forexample an antibody) can be achieved by drawing the appropriate liquidinto the tip and holding it there for a predetermined time. Removal ofsamples and reagents can be achieved by expelling the liquid into areservoir or an absorbent pad in a device as described. Another reagentcan then be drawn into the tip according to instructions or protocolfrom the programmable processor.

A system can comprise a holder or engager for moving the assay units ortips. An engager may comprise a vacuum assembly or an assembly designedto fit snugly into a boss of an assay unit tip. For example, a means formoving the tips can be moved in a manner similar to the fluid transferdevice heads. The device can also be moved on a stage according to theposition of an engager or holder.

In an embodiment, an instrument for moving the tips is the same as aninstrument for moving a volume of sample, such as a fluid transferdevice as described herein. For example, a sample collection tip can befit onto a pipette head according to the boss on the collection tip. Thecollection tip can then be used to distribute the liquid throughout thedevice and system. After the liquid has been distributed, the collectiontip can be disposed, and the pipette head can be fit onto an assay unitaccording to the boss on the assay unit. The assay unit tip can then bemoved from reagent unit to reagent unit, and reagents can be distributedto the assay unit according to the aspiration- or pipette-type actionprovided by the pipette head. The pipette head can also perform mixingwithin a collection tip, assay unit, or reagent unit by aspiration- orsyringe-type action.

In another embodiment, tips containing liquids including assay reactionmixtures can be disconnected from the pipetting device and “parked” atspecific locations within the instrument or within a disposable unit. Ifneeded, tips can be capped using a seal (as used in the centrifuge) toprevent liquids from draining out. In some embodiments, the seal can bea vinyl seal.

Exemplary Sample Tips

A variety of container shapes can be utilized as sample tips, reactionchambers, and cuvettes. For example, a cuvette can be circular,cylindrical, square, rectangular, cubical, conical, pyramidal, or anyother shape capable of holding a sample of fluid. Rectangular cuvetteswhere a light beam impinges at right angles to the cuvette surfaces asshown in plan and section views in FIG. 63 can be employed. In suchrectangular cuvettes, the liquid sample that is illuminated is alsorectangular and is defined by the cuvette. Cuvettes with circularcross-sections can also be used. For example, some types of microtiterplates where the illuminated sample volume is in part defined by thesample meniscus as shown below in plan and section view in FIG. 64.

Variable pathlength cuvettes can be used to optimize and extend theassay response and minimize the volume of sample required to measure theassay. Cuvettes can be longer in relation to their cross-section in atleast one region. In some cases, the pathlength of a cuvette can beselected based on cuvette geometry and/or material. Different cuvettescan be selected for different assays.

In the present invention, one preferred version of the assay cuvette hasa circular cross-section in the direction of the light beam as shown inFIG. 65. The use of a cuvette with a circular cross-section has severaladvantages, including, but not limited to the following:

1. The optical pathlength can be precisely defined. Dimensionalprecision of injection-molded parts have been found to be better than1-2% CV. In conventional microtiter plates the unconstrained liquidmeniscus can introduce imprecision in pathlength.

2. The open-ended character and circular section of the tips confersexcellent fluid handling characteristics, making aspiration of liquidsvery precise.

3. The optical image of the tips provides for the ability to identifythe tip location and boundaries of the liquid column and to locate veryprecisely the center of the tip where the signal is maximal.

4. More than one liquid sample can be incubated and analyzed in the sametip. This is because in the narrow part of the tip, very little materialtransfer occurs (in the axial direction) between adjacent “slugs” ofliquid.

An exemplary tip may have the following general features:

Tip length: 0.5-4 cm

Tip OD: 0.2-1.0 cm

Tip ID: 0.1-0.5 cm

Tip capacity for liquids: 5-50 uL

Tip dimensional precision: generally better than 2% or +/−0.001 cm

Tip configuration: The tip will generally have a feature that engageswith a pipette (cylindrical) so as to form a fluid tight seal. There isa region generally cylindrical or conical which is used for imaging.Generally the optical part of the tip will have at least two differentsections with different pathlengths. The lower end of the tip willtypically be narrow so as to aid in retention of vertical liquid columnsunder gravity

Tip material: Clear or uniformly specular plastic (polystyrene,polypropylene etc.) (transmission of light in the visible >80%)

For imaging purposes, the tip can generally be clear or translucent, butthe tips do not have to be clear to work well as assay cuvettes whenthree-color analysis is used. Tip cuvettes which appear “cloudy” mayfunction similarly to clear tips. The cloudy tips are made in injectionmolds with non-polished or textured surfaces or by adding some lightscattering material to the plastic used to fabricate the tips. The lightscattering intensity of such cloudy tips may be chosen to be not sogreat as to obscure the colored liquid to be measured. In general, theimpact of light scattering on transmitted light can be selected to beless than 10, (20, and 30%) relative to the impact of the coloredmaterial. The light scattering effect can be selected such that thelight scattering of the cloudy tips is uniform.

The tips and reaction chambers described herein can be comprised of acylindrical (or conical) shaft about 2 cm in length and having an innerdiameter of about 1-5 mm corresponding to a capacity of about 10-50 uL.

In one example, at the upper end of the cylinder is a truncatedcylindrical “boss” fluidically connected to the cylinder and adapted soas to be able to engage with the tapered feature of a pipetter. Thelower end of the tip may be narrowed to provide a feature that enablesthe tip to hold its liquid contents when oriented vertically and notattached to the pipetter. The tip may be a pointed tip. The externalshape of the lower end of the tip is typically also somewhat pointedwith the diameter being reduced from the main part of the cylindricalshaft toward the end so as to be capable of being fluidically sealedwith a flexible (vinyl) cap into which the tip end is press fit. Tipsare usually made of molded plastic (polystyrene, polypropylene and thelike). The tips can be clear or translucent such that information aboutthe sample can be acquired by imaging.

FIG. 4, FIG. 5, and FIG. 6 show an example of a tip. The tip isconfigured with (1) an upper feature that can engage to form an airtight seal with a pipette head, (2) a basically cylindrical (actuallyconical with a very slight draft angle) shaft and a narrow, pointedlower tip. This tip can form a liquid-tight seal with a cap. The pointedshape aids in getting good conformance with the cap under moderateforce. The material used is injection-molded polystyrene. The overalldimensions are: 32 mm long, about 7.6 mm largest outer diameter, usefulcapacity about 20 uL. The dimensions of the tip can be scaled to alarger volume. For example, for a 50 uL sample, the IDs can be increasedby about 1.6-fold.

Sealing can be achieved using a cap made of vinyl or other materialswhich is easily press-fit to the narrow end of the sample containmentmeans using force generated by motion of the instrument stage in thez-direction. A bubble of air can become trapped within the tip when thetip is capped. A centrifugation step can be used to drive the bubble tothe top of the column of blood so as to eliminate the effects of thebubble. The dimensions of the tip and/or the dimensions of the tipholder in a centrifuge can be matched such that a tip can be secured forcentrifugation.

Sample Preparation

The invention provides for systems, methods, and devices for theprocessing and analysis of samples can be collected from a variety ofsources. For example, the sample can be collected from patients,animals, or the environment. The sample can be a bodily fluid. Anybodily fluids suspected to contain an analyte of interest can be used inconjunction with the system or devices of the invention. Commonlyemployed bodily fluids include but are not limited to blood, serum,saliva, urine, gastric and digestive fluid, tears, stool, semen, vaginalfluid, interstitial fluids derived from tumorous tissue, andcerebrospinal fluid.

In some embodiments, the bodily fluid is a blood sample from a humanpatient. The blood source can be collected from a finger prick and havea volume of less than about 0.5, 1, 5, 10, 20, 50, 100, 200, 300, 400,500, or 1000 uL.

A bodily fluid may be drawn from a patient and provided to a device in avariety of ways, including but not limited to, lancing, injection, orpipetting.

As used herein, the terms “subject” and “patient” are usedinterchangeably herein, and refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.

In one embodiment, a lancet punctures the skin and withdraws a sampleusing, for example, gravity, capillary action, aspiration, or vacuumforce. The lancet may be part of the device, or part of a system or astand-alone component. Where needed, the lancet may be activated by avariety of mechanical, electrical, electromechanical, or any other knownactivation mechanism or any combination of such methods. In anotherembodiment where no active mechanism is required, a patient can simplyprovide a bodily fluid to the device, as for example, could occur with asaliva sample. The collected fluid can be placed in the samplecollection unit within the device. In yet another embodiment, the devicecomprises at least one microneedle which punctures the skin.

The volume of bodily fluid to be used with a device can be less thanabout 500 microliters, typically between about 1 to 100 microliters.Where desired, a sample of 1 to 50 microliters, 1 to 40 microliters, 1to 30 microliters, 1 to 10 microliters or even 1 to 3 microliters can beused for detecting an analyte using the device. In an embodiment, thevolume of bodily fluid used for detecting an analyte utilizing thesubject devices or systems is one drop of fluid. For example, one dropof blood from a pricked finger can provide the sample of bodily fluid tobe analyzed with a device, system or method described herein.

A sample of bodily fluid can be collected from a subject directly into atip of the described herein, or can be later transferred to a tip.

Sample Dilution

In some instances, the configuration of the processor to direct fluidtransfer effects a degree of dilution of the bodily fluid sample in thearray of assay units to bring signals indicative of the plurality ofanalytes being detected within a detectable range, such that saidplurality of analytes are detectable with said system. In an example,the bodily fluid sample comprises at least two analytes that are presentat concentrations that differ by at least 1, 2, 5, 10, 15, 50, 100, 500,1000, 10,000, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ fold. In an example thebodily fluid sample is a single drop of blood. In an embodiment, theconcentrations of at least two analytes present in a sample differs byup to 10 orders of magnitude (for example, a first analyte is present at0.1 pg/mL and a second analyte is present at 500 ug/mL). In anotherexample, some protein analytes are found at concentrations of greaterthan 100 mg/mL, which can extend the range of interest to about twelveorders of magnitude. In the case of measurement of nucleic acid analytessuch as DNA and RNA using exponential amplification methods such aspolymerase reaction, the number of copies of analyte can be increased bya billion fold prior to measurement.

Where desired, a degree of dilution of the bodily fluid sample can bringthe signals indicative of the at least two analytes within thedetectable range.

As described, the systems and devices herein can enable many features ofthe flexibility of laboratory setting in a POC environment. For example,samples can be collected and manipulated automatically in a table topsize or smaller device or system. A common issue in POC devices isachieving different dilution ranges when conducting a plurality ofassays, wherein the assays may have significantly different sensitivityor specificity. For example, there may be two analytes in a sample, butone analyte has a high concentration in the sample and the other analytehas a very low concentration. As provided, the systems and devicesherein can dilute the sample to significantly different levels in orderto detect both analytes. Alternatively, a sample may be split into twoor more samples, which may enable individual analytes to be detected atvarious levels of dilution.

For example, if the analyte is in a high concentration, a sample can beserially diluted to the appropriate detection range and provided to acapture surface for detection. In the same system or device, a samplewith an analyte in a low concentration may not need to be diluted. Inthis manner, the assay range of the POC devices and systems providedherein can be expanded from many of the current POC devices.

In POC assay systems using disposable cartridges containing the diluentthere is often a practical limit to the extent of dilution. For example,if a small blood sample is obtained by fingerstick (for example, about20 microliters) is to be diluted and the maximum volume of diluent thatcan be placed in a tube is 250 microliters, the practical limit ofdilution of the whole sample is about 10-fold. In an example herein, asystem can aspirate a smaller volume of the sample (for example about 2microliters) making the maximum dilution factor about 100-fold. For manyassays, such dilution factors are acceptable but for an assay like thatof CRP (as described in the examples herein) there is a need to dilutethe sample much more. Separation-based ELISA assays can have anintrinsic limitation in the capacity of the capture surface to bind theanalyte (for example about a few hundred ng/ml for a typical proteinanalyte). Some analytes are present in blood at hundreds ofmicrograms/ml. Even when diluted by 100-fold, the analyte concentrationmay be outside the range of calibration. In an exemplary embodiment of asystem, device, and fluid transfer device herein, multiple dilutions canbe achieved by performing multiple fluid transfers of the diluent intoan individual assay unit or sample collection unit. For example, if theconcentration of an analyte is very high in a sample as described above,the sample can be diluted multiple times until the concentration of theanalyte is within an acceptable detection range. The systems and methodsherein can provide accurate measurements or estimations of the dilutionsin order to calculate the original concentration of the analyte.

Sample Separation

In some embodiments of the invention, a sample can be prepared foranalysis by an initial separation step. For example, if the assay is toanalyze DNA, a DNA separation step can be employed to eliminate orreduce contaminants or unwanted source material. The separation step canutilize chromatography, centrifugation, liquid-liquid extraction,solid-liquid extraction, affinity binding, or any other mechanisms knownto one skilled in the art.

In some embodiments, a blood sample to be analyzed is first processed byseparating the plasma component from the blood sample. This step can beperformed using a variety of techniques, such as filtration,centrifugation, and affinity binding. Centrifugation can be an efficientmethod for separation of blood sample components, and can be employed inthe present invention.

Plasma Separation

Blood can be introduced into a close ended or sealable tip in a varietyof ways, for example samples can be provided in a tube and a sealabletip can receive the sample from the tube via capillary action or viapneumatic force. One preferred means of introduction is the use ofcapillary action. Alternatively, container used to hold the sample forcentrifugal separation can be configured with only one opening as in aconventional centrifuge.

The tip, once filled with blood, can be moved automatically to alocation in a disposable cartridge where there is a sealing element. Thesealing element can be a small “cup” made of a deformable (pliant)material (vinyl, silicone or the like) conformed to fit on the lower endof the tip and to seal it. The tip is pressed into the seal by theinstrument thus forming a liquid-tight junction. The sealed tip is thenmoved to a small centrifuge (typically located in and forming part ofthe instrument) and press-fit into a positioning feature in thecentrifuge rotor such that the lower (sealed) end of the tip butts up toa rigid shelf that will support the tip during the centrifugation step.

The centrifuge rotor can be circular having about 10 cm in diameter. Themass of the blood-containing tip is either (1) small relative to therotor or (2), where desired, balanced by a counter weight located on theopposite part of the rotor such that any vibration during thecentrifugation step is minimized One exemplary orientation of thecentrifuge rotor is vertical (axis of rotation horizontal). The rotor ismounted in a drive shaft with is driven by an electric motor.

Centrifugation can be achieved by spinning the rotor at about 15,000 rpmfor 5 minutes. During this process, the particular elements in the blood(red cells and white cells) sediment to the sealed end of the tip andform a closely packed column with cell free plasma separated at the partof the tip distal from the seal.

The tip containing the separated sample can then be placed vertically ina location accessible to a fluid handling device comprised of a narrowpipette tip (“sample acquisition tip”) mounted on a pipetting device inturn mounted on an x-y-z stage.

Plasma can now be efficiently recovered from the centrifuged sample.This is achieved by moving the sample acquisition tip vertically alongthe axis of the centrifuge tip so that it comes into fluid contact withthe plasma and can draw the plasma upwards using, e.g., pneumatic means.

Optionally, this operation can be monitored using a camera or otherimaging device which can be used both to measure the sample hematocritand to provide information as to the location of the plasma/red cellboundary to the stage/pipetter controller. With the aid of imaging theseparated blood, a narrow pipette tip fitted to a pipette is slowlymoved vertically down, such that the tip is directed axially down thesample containment means until it contacts the plasma. The tip is thenmoved further until it is close (within less than about 3, 2, 1, 0.5, or0.1 mm) of the packed cell interface. At the same time, plasma isaspirated into the narrow pipette tip under computer control. The plasmacan be aspirated simultaneously while moving the narrow pipette tip intothe plasma column so that the plasma does not become displaced into theupper part of the sample containment means. The aspiration can becontrolled to avoid air being aspirated during the plasma removal step.

In general, a pipette tip with a very narrow end, such as those used toapply samples to an electrophoresis system, can be used to aspirate theplasma from the centrifuged sample tip. The narrow tip is typicallyconical or tapered and has dimensions 1-3×0.1-0.5 cm (length×diameter)and made of any of a variety of materials (polypropylene, polystyreneetc.). The material can be clear or translucent in the visible. One endof the tip engages with a pipetting device. The other is very narrow(0.05-0.5 mm OD) such that it can move into the sample tip withouttouching the inner surface of the sample tip. Even if there is contactbetween the plasma aspiration tip and the sample tip, plasma aspirationis not hindered.

A schematic of the plasma aspiration process at the stage where theplasma aspiration tip is located just above the plasma-packed cellinterface during the aspiration step is shown in FIG. 7.

In this way we have found that almost all of the plasma can be removedleaving as little as e.g. 1 uL in the centrifuged sample tip. Thiscorresponds to about 11 uL of plasma (90% recovery) from 20 uL of bloodwith a 40% hematocrit. Additionally the quality of the plasma sample(with respect to hemolysis, lipemia and icteria) can be determined froman image of the centrifuged sample.

The aspirated plasma can be moved to other locations for dilution andmixing with assay reagents so that assays for analytes industry but notlimited to metabolites, electrolytes, protein biomarkers, drugs andnucleic acids may be performed.

Separation of White Blood Cells

A further use of the invention is to isolate and concentrate the whitecells from blood. In one aspect of the invention, the blood sample isfirst subject to a process which lyses the red cells (and optionallyfixes the white cells) by adding a reagent (For example, BD Pharmlyse™555899 or BD FACS™ Lysing Solution 349202) to the blood and mixing.Following a brief incubation, the lysed sample is subject tocentrifugation as described above such that the white cells areconcentrated at the sealed end of the blood tip. The lysed red cellsolution can then be removed by aspiration. Recovery of the white cellsis achieved by either (1) addition of a small amount of a buffersolution and repeated up and down aspiration to re-suspend the cellsfollowed by displacement into a receptacle or (2) removal of the sealand downward displacement of the packed cells into a receptacle usingair pressure.

An alternate scheme allows recovery of white cells without lysis of thered cells. After centrifugation of blood (as is well known) the whitecells form a layer on top of the packed red cells known as the BuffyCoat. Following removal of most of the plasma (as above) the white cellscan be efficiently recovered by (1) optionally adding a small volume(e.g. about 5 uL) of isotonic buffer, or (2) using the residual plasmaand re-suspending the white cells by repeated aspiration and/ormechanical stirring using the sample acquisition tip. Once suspended,the resulting mixture of white cells together with a small proportion ofred cells also re-suspended can be acquired by aspiration for analysisof the white cells. In this way most of the white cells (typically all)can be recovered with only a small (contaminating) quantity of red cells(typically less than 5% of the original).

Centrifuges

FIG. 1, FIG. 2, and FIG. 3 show scale perspectives of a centrifuge (FIG.1—side view, FIG. 2—front face view, FIG. 3—rear view) that can beintegrated into the system. The centrifuge may contain an electric motorcapable of turning the rotor at 15,000 rpm. One type of centrifuge rotoris shaped somewhat like a fan blade is mounted on the motor spindle in avertical plane. Affixed to the rotor is an element which holds thesample holding means (tip) and provides a ledge or shelf on which theend of the tip distal to the motor axis rests and which provides supportduring the centrifugation so that the sample cannot escape. The tip maybe further supported at its proximal end by a mechanical stop in therotor. This can be provided so that the force generated duringcentrifugation does not cause the tip to cut through the soft vinyl cap.The tip can be inserted and removed by standard pick and placemechanisms but preferably by a pipette. The rotor is a single piece ofacrylic (or other material) shaped to minimize vibration and noiseduring operation of the centrifuge. The rotor is (optionally) shaped sothat when it is oriented in particular angles to the vertical, othermovable components in the instrument can move past the centrifuge. Thesample holding means are centrifugally balanced by counter masses on theopposite side of the rotor such that the center of rotational inertia isaxial relative to the motor. The centrifuge motor may provide positionaldata to a computer which can then control the rest position of the rotor(typically vertical before and after centrifugation).

As may be seen from the two graphs in FIG. 8 and FIG. 9, to minimizecentrifugation time (without generating too much mechanical stressduring centrifugation) according to published standards (DIN 58933-1;for the U.S. the CLSI standard H07-A3 “Procedure for Determining PackedCell Volume by the Microhematocrit Method”; Approved Standard—ThirdEdition) convenient dimensions for the rotor are in the range of about5-10 cm spinning at about 10-20 thousand rpm giving a time to pack thered cells of about 5 min.

An exemplary equation for calculating centrifugation force is shownbelow:

${RGF} - \frac{{T\left( {2\pi\; N} \right)}^{2}}{g}$

Where:

g is earth's gravitational acceleration,

τ is the rotational radius,

N is the rotational speed, measured in revolutions per unit of time.

Where:

τcm is the rotational radius measured in centimeters (cm),

N_(RPM) is rotational speed measured in revolutions per minute (RPM).

RCF=1.118×10⁻⁶τ_(cm) N _(RPM) ²

In some embodiments, a centrifuge may be a horizontally orientedcentrifuge with a swinging bucket design. In some preferableembodiments, the axis of rotation of the centrifuge is vertical. Inalternate embodiments, the axis of rotation can be horizontal or at anyangle. The centrifuge may be capable of simultaneously spinning two ormore vessels and may be designed to be fully integrated into anautomated system employing computer-controlled pipettes. In someembodiments, the vessels may be close-bottomed. The swinging bucketdesign may permit the centrifugation vessels to be passively oriented ina vertical position when stopped, and spin out to a fixed angle whenspinning. In some embodiments, the swinging buckets may permit thecentrifugation vessels to spin out to a horizontal orientation.Alternatively they may spin out to any angle between a vertical andhorizontal position (e.g., about 15, 30, 45, 60, or 75 degrees fromvertical. The centrifuge with swinging bucket design may meet thepositional accuracy and repeatability requirements of a robotic system anumber of positioning systems are employed.

A computer-based control system may use position information from anoptical encoder in order to spin the rotor at controlled slow speeds.Because an appropriate motor could be designed for high-speedperformance, accurate static positions need not be held using positionfeedback alone. In some embodiments, a cam in combination with asolenoid-actuated lever may be employed to achieve very accurate andstable stopping at a fixed number of positions. Using a separate controlsystem and feedback from Hall-Effect sensors built into the motor, thevelocity of the rotor can be very accurately controlled at high speeds.

Because a number of sensitive instruments must function simultaneouslywithin the assay instrument system, the design of the centrifugepreferably minimizes or reduces vibration. The rotor may beaerodynamically designed with a smooth exterior—fully enclosing thebuckets when they are in their horizontal position. Also, vibrationdampening can be employed in multiple locations in the design of thecase.

Rotor

A centrifuge rotor can be a component of the system which may hold andspin the centrifugation vessel(s). The axis of rotation can be vertical,and thus the rotor itself can be positioned horizontally. However, inalternate embodiments, different axes of rotation and rotor positionscan be employed. There are two components known as buckets positionedsymmetrically on either side of the rotor which hold the centrifugationvessels. Alternative configurations are possible in which buckets areoriented with radial symmetry, for example three buckets oriented at 120degrees. Any number of buckets may be provided, including but notlimited to 1, 2, 3, 4, 5, 6, 7, 8, or more buckets. The buckets can beevenly spaced from one another. For example, if n buckets are providedwhere n is a whole number, then the buckets may be spaced about 360/ndegrees apart from one another. In other embodiments, the buckets neednot be spaced evenly around one another or with radial symmetry.

When the rotor is stationary, these buckets, influenced by gravity, maypassively fall such as to position the vessels vertically and to makethem accessible to the pipette. FIG. 111 shows an example of a rotor atrest with buckets vertical. In some embodiments, the buckets maypassively fall to a predetermined angle that may or may not be vertical.When the rotor spins, the buckets are forced into a nearly horizontalposition or to a predetermined angle by centrifugal forces. FIG. 112shows an example of a rotor at a speed with buckets at a small angle tohorizontal. There can be physical hard stops for both the vertical andhorizontal positions acting to enforce their accuracy and positionalrepeatability.

The rotor may be aerodynamically designed with a disk shape, and as fewphysical features as possible in order to minimize vibration caused byair turbulence. To achieve this, the outer geometry of the bucket mayexactly match that of the rotor such that when the rotor is spinning andthe bucket can be forced horizontal the bucket and rotor can beperfectly aligned.

To facilitate plasma extraction, the rotor may be angled down toward theground relative to the horizon. Because the angle of the bucket can bematched to that of the rotor, this may enforce a fixed spinning anglefor the bucket. The resulting pellet from such a configuration could beangled relative to the vessel when placed upright. A narrow extractiontip may be used to aspirate plasma from the top of the centrifugationvessel. By placing the extraction tip near the bottom of the slopecreated by the angle pellet, the final volume of plasma can be moreefficiently extracted without disturbing the sensitive buffy coat.

A variety of tubes designs can be accommodated in the buckets of thedevice. In some embodiments, the various tube designs may be closedended. Some are shaped like conventional centrifuge tubes with conicalbottoms. Other tube designs may be cylindrical. Tubes with a low ratioof height to cross-sectional area may be favored for cell processing.Tubes with a large ratio (>10:1) may be suitable for accuratemeasurement of hematocrit and other imaging requirements. However, anyheight to cross-sectional area ratio may be employed. The buckets can bemade of any of several plastics (polystyrene, polypropylene), or anyother material discussed elsewhere herein. Buckets have capacitiesranging from a few microliters to about a milliliter. The tubes may beinserted into and removed from the centrifuge using a “pick and place”mechanism.

Control System

Due to the spinning and positioning requirements of the centrifugedevice, a dual control system approach may be used. To index the rotorto specific rotational orientations, a position based control system maybe implemented. In some embodiments, the control system may employ a PID(Proportional Integral Derivative) control system. Other feedbackcontrol systems known in the art can be employed. Positional feedbackfor the position controller may be provided by a high-resolution opticalencoder. For operating the centrifuge at low to high speeds, a velocitycontroller may be implemented, while employing a PID control systemtuned for velocity control. Rotational rate feedback for the velocitycontroller may be provided by a set of simple Hall-Effect sensors placedon the motor shaft. Each sensor may generate a square wave at one cycleper motor shaft rotation.

Stopping Mechanism

To consistently and firmly position the rotor in a particular position,a physical stopping mechanism may be employed. In one embodiment, thestopping mechanism may use a cam, coupled to the rotor, along with asolenoid-actuated lever. The cam may be shaped like a circular disk witha number of “C” shaped notches machined around the perimeter. Toposition the centrifuge rotor, its rotational velocity may first belowered to, at most, 30 RPM. In other embodiments, the rotationalvelocity may be lowered to any other amount, including but not limitedto about 5 rpm, 10 rpm, 15 rpm, 20 rpm, 25 rpm, 35 rpm, 40 rpm, or 50rpm. Once the speed is sufficiently slow, the lever may be actuated. Atthe end of the lever is a cam follower which may glide along theperimeter of the cam with minimal friction. Once the cam followerreaches the center of a particular notch in the cam, the force of thesolenoid-actuated lever can overcome that of the motor and the rotor maybe brought to a halt. At that point the motor may be electronicallybraked, and, in combination with the stopping mechanism a rotationalposition can be very accurately and firmly held indefinitely.

Centrifuge Buckets

The centrifuge swing-out buckets may be configured to accommodatedifferent type of centrifuge tubes. In preferable embodiments, thevarious tube types may have a collar or flange at their upper (open)end. This collar or flange feature may rests on the upper end of thebucket and support the tube during centrifugation. As shown in FIGS.113, 114, and 115, conical and cylindrical tubes of various lengths andvolumes can be accommodated. FIGS. 113, 114, and 115 provide examples ofbuckets and other bucket designs may be employed. For example, FIG. 113,shows an example of a bucket configuration. The bucket may have sideportions that mate with the centrifuge and allow the bucket to swingfreely. The bucket may have a closed bottom and an opening at the top.FIG. 114 shows an example of a centrifugation vessel mated with thebucket. As previously mentioned, the bucket may be shaped to acceptvarious configurations of centrifugation vessels. The centrifugationvessel may have one or more protruding member that may rest upon thebucket. The centrifugation vessel may be shaped with one or more featurethat may mate with the centrifugation bucket. The feature may be ashaped feature of the vessel or one or more protrusion. FIG. 115 showsan example of another centrifugation vessel that can be mated with thebucket. As previously described, the bucket can have one or more shapedfeature that may allow different configurations of centrifugationvessels to mate with the bucket.

Centrifuge Tubes and Sample Extraction Means:

The centrifuge tube and extraction tip may be provided individually andcan be mated together for extraction of material followingcentrifugation. The centrifugation tube and extraction tip may bedesigned to deal with complex processes in an automated system. FIG. 116shows an example of a centrifugation vessel. FIG. 117 shows an exampleof an extraction tip. FIG. 118 provides an example of how thecentrifugation vessel and extraction tip may mate. Any dimensions areprovided by way of example only, and other dimensions of the same ordiffering proportions may be utilized

The system can enable one or more of the following:

-   -   1. Rapid processing of small blood samples (typically 5-50 uL)    -   2. Accurate and precise measurement of hematocrit    -   3. Efficient removal of plasma    -   4. Efficient re-suspension of formed elements (red and white        blood cells)    -   5. Concentration of white cells (following labeling with        fluorescent antibodies and fixation plus lysis of red cells)    -   6. Optical confirmation of red cell lysis and recovery of white        cells

Centrifugation Vessel and Extraction Tip Overview

A custom vessel and tip may be used for the operation of the centrifugein order to satisfy the variety of constraints placed on the system. Thecentrifugation vessel may be a closed bottom tube designed to be spun inthe centrifuge. In some embodiments, the centrifugation vessel may bethe vessel illustrated in FIG. 116 or may have one or more featuresillustrated in FIG. 116. It may have a number of unique featuresenabling the wide range of required functionality including hematocritmeasurement, RBC lysing, pellet re-suspension and efficient plasmaextraction. The extraction tip may be designed to be inserted into thecentrifugation vessel for precise fluid extraction, and pelletre-suspension. In some embodiments, the extraction tip may be the tipillustrated in FIG. 117 or may have one or more features illustrated inFIG. 117. Exemplary specifications for each tip are discussed herein.

Centrifugation Vessel

The centrifugation vessel may be designed to handle two separate usagescenarios, each associated with a different anti-coagulant and wholeblood volume.

A first usage scenario may require that 40 uL of whole blood withHeparin be pelleted, the maximum volume of plasma be recovered, and thehematocrit measured using computer vision. In the case of 60% hematocritor below the volume of plasma required or preferable may be about 40uL*40%=16 uL.

In some embodiments, it will not be possible to recover 100% of theplasma because the buffy coat must not be disturbed, thus a minimumdistance must be maintained between the bottom of the tip and the top ofthe pellet. This minimum distance can be determined experimentally butthe volume (V) sacrificed as a function of the required safety distance(d) can be estimated using: V(d)=d*π1.25 mm². For example, for arequired safety distance of 0.25 mm, the sacrificed volume could be 1.23uL for the 60% hematocrit case. This volume can be decreased bydecreasing the internal radius of the hematocrit portion of thecentrifugation vessel. However, because in some embodiments, that narrowportion must fully accommodate the outer radius of the extraction tipwhich can be no smaller than 1.5 mm, the existing dimensions of thecentrifugation vessel may be close to the minimum

Along with plasma extraction, in some embodiments it may also berequired that the hematocrit be measured using computer vision. In orderto facilitate this process the total height for a given volume ofhematocrit may be maximized by minimizing the internal diameter of thenarrow portion of the vessel. By maximizing the height, the relationshipbetween changes in hematocrit volume and physical change in columnheight may be optimized, thus increasing the number of pixels that canbe used for the measurement. The height of the narrow portion of thevessel may also be long enough to accommodate the worst-case scenario of80% hematocrit while still leaving a small portion of plasma at the topof the column to allow for efficient extraction. Thus, 40 uL*80%=32 uLmay be the required volume capacity for accurate measurement of thehematocrit. The volume of the narrow portion of the tip as designed maybe about 35.3 uL which may allow for some volume of plasma to remain,even in the worst case.

A second usage scenario is much more involved, and may require one,more, or all of the following:

-   -   whole blood pelleted    -   plasma extracted    -   pellet re-suspended in lysing buffer and stain    -   remaining white blood cells (WBCs) pelleted    -   supernatant removed    -   WBCs re-suspended    -   WBC suspension fully extracted

In order to fully re-suspend a packed pellet, experiments have shown onecan physically disturb the pellet with a tip capable of completelyreaching the bottom of the vessel containing the pellet. A preferablegeometry of the bottom of the vessel using for re-suspension seems to bea hemispherical shape similar to standard commercial PCR tubes. In otherembodiments, other vessel bottom shapes may be used. The centrifugationvessel, along with the extraction tip, may be designed to facilitate there-suspension process by adhering to these geometrical requirementswhile also allowing the extraction tip to physically contact the bottom.

During manual re-suspension experiments it was noticed that physicalcontact between the bottom of the vessel, and the bottom of the tip maycreate a seal that prohibits fluid movement. A delicate spacing may beused in order to both fully disturb the pellet, while allowing fluidflow. In order to facilitate this process in a robotic system, aphysical feature may be added to the bottom of the centrifugationvessel. In some embodiments, this feature may comprise four smallhemispherical nubs placed around the perimeter of the bottom portion ofthe vessel. When the extraction tip is fully inserted into the vesseland allowed to make physical contact, the end of the tip may rest on thenubs, and fluid is allowed to freely flow between the nubs. This mayresult in a small amount of volume (˜0.25 uL) lost in the gaps.

During the lysing process, in some implementations, the maximum expectedfluid volume is 60 uL, which, along with 25 uL displaced by theextraction tip may demand a total volume capacity of 85 uL. A designwith a current maximum volume of 100 uL may exceed this requirement.Other aspects of the second usage scenario require similar or alreadydiscussed tip characteristics.

The upper geometry of the centrifugation vessel may be designed to matewith a pipette nozzle. Any pipette nozzle described elsewhere herein orknown in the art may be used. The external geometry of the upper portionof the vessel may exactly match that of a reaction tip which both thecurrent nozzle and cartridge may be designed around. In someembodiments, a small ridge may circumscribe the internal surface of theupper portion. This ridge may be a visual marker of the maximum fluidheight, meant to facilitate automatic error detection using computervision system.

In some embodiments, the distance from the bottom of the fully matednozzle to the top of the maximum fluid line is 2.5 mm. This distance is1.5 mm less than the 4 mm recommended distance adhered to by theextraction tip. This decreased distance may be driven by the need tominimize the length of the extraction tip while adhering to minimumvolume requirements. The justification for this decreased distance stemsfrom the particular use of the vessel. Because, in some implementations,fluid may be exchanged with the vessel from the top only, the maximumfluid it will ever have while mated with the nozzle is the maximumamount of whole blood expected at any given time (40 uL). The height ofthis fluid may be well below the bottom of the nozzle. Another concernis that at other times the volume of fluid in the vessel may be muchgreater than this and wet the walls of up to the height of the nozzle.In some embodiments, it will be up to those using the vessel to ensurethat the meniscus of any fluids contained within the vessel do notexceed the max fluid height, even if the total volume is less than themaximum specified. In other embodiments, other features may be providedto keep the fluid contained within the vessel.

Any dimensions, sizes, volumes, or distances provided herein areprovided by way of example only. Any other dimension, size, volume ordistance may be utilized which may or may not be proportional to theamounts mentioned herein.

The centrifugation vessel can be subjected to a number of forces duringthe process of exchanging fluids and rapidly inserting and removingtips. If the vessel is not constrained, it is possible that these forceswill be strong enough to lift or otherwise dislodge the vessel from thecentrifuge bucket. In order to prevent movement, the vessel should besecured in some way. To accomplish this, a small ring circumscribing thebottom exterior of the vessel was added. This ring can easily be matedwith a compliant mechanical feature on the bucket. As long as theretaining force of the nub is greater than the forces experienced duringfluid manipulations, but less than the friction force when mated withthe nozzle then the problem is solved.

Extraction Tip

The Extraction Tip may be designed to interface with the centrifugationvessel, efficiently extracting plasma, and re-suspending pelleted cells.Where desired, its total length (e.g., 34.5 mm) may exactly match thatof another blood tip including but not limited to those described inU.S. Ser. No. 12/244,723 (incorporated herein by reference) but may belong enough to physically touch the bottom of the centrifugation vessel.The ability to touch the bottom of the vessel may be required in someembodiments, both for the re-suspension process, and for completerecovery of the white cell suspension.

The required volume of the extraction tip may be determined by themaximum volume it is expected to aspirate from the centrifugation vesselat any given time. In some embodiments, this volume may be approximately60 uL, which may be less than the maximum capacity of the tip which is85 uL. In some embodiments, a tip of greater volume than required volumemay be provided. As with the centrifugation vessel, an internal featurecircumscribing the interior of the upper portion of the tip may be usedto mark the height of this maximum volume. The distance between themaximum volume line and the top of the mated nozzle may be 4.5 mm, whichmay be considered a safe distance to prevent nozzle contamination. Anysufficient distance to prevent nozzle contamination may be used.

The centrifuge may be used to sediment precipitated LDL-cholesterol.Imaging may be used to verify that the supernatant is clear, indicatingcomplete removal of the precipitate.

In one example, plasma may be diluted (e.g., 1:10) into a mixture ofdextran sulfate (25 mg/dL) and magnesium sulfate (100 mM), and may bethen incubated for 1 minute to precipitate LDL-cholesterol. The reactionproduct may be aspirated into the tube of the centrifuge, capped thenand spun at 3000 rpm for three minutes. FIGS. 119, 120, and 121 areimages that were taken of the original reaction mixture prior tocentrifugation (showing the white precipitate), following centrifugation(showing a clear supernatant) and of the LDL-cholesterol pellet (afterremoval of the cap), respectively.

Other examples of centrifuges that can be employed in the presentinvention are described in U.S. Pat. Nos. 5,693,233, 5,578,269,6,599,476 and U.S. Patent Publication Nos. 2004/0230400, 2009/0305392,and 2010/0047790, which are incorporated by reference in their entiretyfor all purposes.

Example Protocols

Many variations of protocol may be used for centrifugation andprocessing. For example, a typical protocol for use of the centrifuge toprocess and concentrate white cells for cytometry may include one ormore of the following steps. The steps below may be provided in varyingorders or other steps may be substituted for any of the steps below:

-   -   1. Receive 10 uL blood anti-coagulated with EDTA (pipette        injects the blood into the bottom of the centrifuge bucket)    -   2. Sediment the red and white cells by centrifugation (<5        min×10,000 g).    -   3. Measure hematocrit by imaging    -   4. Remove plasma slowly by aspiration into the pipette (4 uL        corresponding to the worst case scenario [60% hematocrit])        without disturbing the cell pellet.    -   5. Re-suspend the pellet after adding 20 uL of an appropriate        cocktail of up to five fluorescently labeled antibodies¹        dissolved in buffered saline+BSA (1 mg/mL) (total reaction        volume about 26 uL²). ¹ Concentration will be adjusted        appropriately to deal with the different volume ratio relative        to standard laboratory method (specifically about 5×lower)² If        necessary, this volume can be bigger to have optimal staining        but not more than 50 uL.    -   6. Incubate for 15 minutes at 37 C.    -   7. Prepare lysing/fixative reagent by mixing red cell lysing        solution (ammonium chloride/potassium bicarbonate) with white        cell fixative reagent (formaldehyde).    -   8. Add 30 uL lysing/fixative reagent (total reaction volume        about 60 uL).    -   9. Incubate 15 minutes at 37 C    -   10. Sediment the white cells by centrifugation (5 min, 10,000        g).    -   11. Remove the supernatant hemolysate (about 57 uL).    -   12. Re-suspend the white cells by adding 8 uL buffer (isotonic        buffered saline).    -   13. Measure the volume accurately.    -   14. Deliver sample (c 10 uL) to cytometry.

The steps may include receiving a sample. The sample may be a bodilyfluid, such as blood, or any other sample described elsewhere herein.The sample may be a small volume, such as any of the volume measurementsdescribed elsewhere herein. In some instances, the sample may have ananti-coagulant.

A separation step may occur. For example, a density-based separation mayoccur. Such separation may occur via centrifugation, magneticseparation, lysis, or any other separation technique known in the art.In some embodiments, the sample may be blood, and the red and whiteblood cells may be separated.

A measurement may be made. In some instances, the measurement may bemade via imaging, or any other detection mechanism described elsewhereherein. For example, the hematocrit of a separated blood sample may bemade by imaging. Imaging may occur via a digital camera or any otherimage capture device described herein.

One or more component of a sample may be removed. For example, if thesample is separated into solid and liquid components, the liquidcomponent may be moved. The plasma of a blood sample may be removed. Insome instances, the liquid component, such as plasma, may be removed viaa pipette. The liquid component may be removed without disturbing thesolid component. The imaging may aid in the removal of the liquidcomponent, or any other selected component of the sample. For example,the imaging may be used to determine where the plasma is located and mayaid in the placement of the pipette to remove the plasma.

In some embodiments, a reagent or other material may be added to thesample. For example, the solid portion of the sample may be resuspended.A material may be added with a label. One or more incubation step mayoccur. In some instances, a lysing and/or fixative reagent may be added.Additional separation and/or resuspending steps may occur. As needed,dilution and/or concentration steps may occur.

The volume of the sample may be measured. In some instances, the volumeof the sample may be measured in a precise and/or accurate fashion. Thevolume of the sample may be measured in a system with a low coefficientof variation, such as coefficient of variation values describedelsewhere herein. In some instances, the volume of the sample may bemeasured using imaging. An image of the sample may be captured and thevolume of the sample may be calculated from the image.

The sample may be delivered to a desired process. For example, thesample may be delivered for cytometry.

In another example, a typical protocol that may or may not make use ofthe centrifuge for nucleic acid purification may include one or more ofthe following steps. The system may enable DNA/RNA extraction to delivernucleic acid template to exponential amplification reactions fordetection. The process may be designed to extract nucleic acids from avariety of samples including, but not limited to whole blood, serum,viral transfer medium, human and animal tissue samples, food samples,and bacterial cultures. The process may be completely automated and mayextract DNA/RNA in a consistent and quantitative manner. The steps belowmay be provided in varying orders or other steps may be substituted forany of the steps below:

1. Sample Lysis. Cells in the sample may be lysed using achaotropic-salt buffer. The chaotropic-salt buffer may include one ormore of the following: chaotropic salt such as, but not limited to, 3-6M guanidine hydrochloride or guanidinium thiocyanate; sodium dodecylsulfate (SDS) at a typical concentration of 0.1-5% v/v;ethylenediaminetetraacetic acid (EDTA) at a typical concentration of 1-5mM; lysozyme at a typical concentration of 1 mg/mL; proteinase-K at atypical concentration of 1 mg/mL; and pH may be set at 7-7.5 using abuffer such as HEPES. In some embodiments, the sample may be incubatedin the buffer at typical temperature of 20-95° C. for 0-30 minutes.Isopropanol (50%-100% v/v) may be added to the mixture after lysis.

2. Surface Loading. Lysed sample may be exposed to a functionalizedsurface (often in the form of a packed bed of beads) such as, but notlimited to, a resin-support packed in a chromatography style column,magnetic beads mixed with the sample in a batch style manner, samplepumped through a suspended resin in a fluidized-bed mode, and samplepumped through a closed channel in a tangential flow manner over thesurface. The surface may be functionalized so as to bind nucleic acids(e.g. DNA, RNA, DNA/RNA hybrid) in the presence of the lysis buffer.Surface types may include silica, and ion-exchange functional groupssuch as diethylaminoethanol (DEAE). The lysed mixture may be exposed tothe surface and nucleic acids bind.

3. Wash. The solid surface is washed with a salt solution such as 0-2 Msodium chloride and ethanol (20-80% v/v) at pH 7.0-7.5. The washing maybe done in the same manner as loading.

4. Elution. Nucleic acids may be eluted from the surface by exposing thesurface to water or buffer at pH 7-9. Elution may be performed in thesame manner as loading.

Many variations of these protocols or other protocols may be employed bythe system. Such protocols may be used in combination or in the place ofany protocols or methods described herein.

In some embodiments, it is important to be able to recover the cellspacked and concentrated by centrifugation for cytometry. In someembodiments, this may be achieved by use of the pipetting device.Liquids (typically isotonic buffered saline, a lysing agent, a mixtureof a lysing agent and a fixative or a cocktail of labeled antibodies inbuffer) may be dispensed into the centrifuge bucket and repeatedlyaspirated and re-dispensed. The tip of the pipette may be forced intothe packed cells to facilitate the process. Image analysis aids theprocess by objectively verifying that all the cells have beenre-suspended.

Use of the Pipette and Centrifuge to Process Samples Prior to Analysis:

In accordance with an embodiment of the invention, the system may havepipetting, pick-and-place and centrifugal capabilities. Suchcapabilities may enable almost any type of sample pretreatment andcomplex assay procedures to be performed efficiently with very smallvolumes of sample.

Specifically, the system may enables separation of formed elements (redand white cells) from plasma. The system may also enable re-suspensionof formed elements. In some embodiments, the system may enableconcentration of white cells from fixed and hemolysed blood. The systemmay also enable lysis of cells to release nucleic acids. In someembodiments, purification and concentration of nucleic acids byfiltration through tips packed with (typically beaded) solid phasereagents (e.g. silica) may be enabled by the system. The system may alsopermit elution of purified nucleic acids following solid phaseextraction. Removal and collection of precipitates (for exampleLDL-cholesterol precipitated using polyethylene glycol) may also beenabled by the system.

In some embodiments, the system may enable affinity purification. Smallmolecules such as vitamin-D and serotonin may be adsorbed onto beaded(particulate) hydrophobic substrates, then eluted using organicsolvents. Antigens may be provided onto antibody-coated substrates andeluted with acid. The same methods can be used to concentrate analytesfound at low concentrations such as thromboxane-B2 and6-keto-prostaglandin F1α. Antigens may be provided onto antibody oraptamer-coated substrates and then eluted.

In some embodiments, the system may enable chemical modification ofanalytes prior to assay. To assay serotonin (5-Hydroxytryptamine) forexample, it may be required to convert the analyte to a derivative (suchas an acetylated form) using a reagent (such as acetic anhydride). Thismay be done to produce a form of the analyte that can be recognized byan antibody.

Liquids can be moved using the pipette (vacuum aspiration and pumping).The pipette may be limited to relatively low positive and negativepressures (approximately 0.1-2.0 atmospheres). A centrifuge can be usedto generate much higher pressures when needed to force liquids throughbeaded solid phase media. For example, using a rotor with a radius of 5cm at a speed of 10,000 rpm, forces of about 5,000×g (about 7atmospheres) may be generated, sufficient to force liquids throughresistive media such as packed beds. Any of the centrifuge designs andconfigurations discussed elsewhere herein or known in the art may beused.

Measurement of hematocrit with very small volumes of blood may occur.For example, inexpensive digital cameras are capable of making goodimages of small objects even when the contrast is poor. Making use ofthis capability, the system of the present invention may enableautomated measurement of hematocrit with a very small volume of blood.

For example, 1 uL of blood may be drawn into a microcap glass capillary.The capillary may then be sealed with a curable adhesive and thensubject to centrifugation at 10,000×g for 5 minutes. The packed cellvolume may be easily measured and the plasma meniscus (indicated by anarrow) may also be visible so hematocrit can be accurately measured.This may enable the system to not waste a relatively large volume ofblood to make this measurement. In some embodiments, the camera may beused “as is” without operation with a microscope to make a larger image.In other embodiments, a microscope or other optical techniques may beused to magnify the image. In one implementation, the hematocrit wasdetermined using the digital camera without additional opticalinterference, and the hematocrit measured was identical to thatdetermined by a conventional microhematocrit laboratory method requiringmany microliters of sample. In some embodiments, the length of thesample column and of that of the column of packed red cells can bemeasured very precisely (+/−<0.05 mm). Given that the blood samplecolumn may be about 10-20 mm, the standard deviation of hematocrit maybe much better than 1% matching that obtained by standard laboratorymethods.

The system may enable measurement of erythrocyte sedimentation rate(ESR). The ability of digital cameras to measure very small distancesand rates of change of distances may be exploited to measure ESR. In oneexample, three blood samples (15 uL) were aspirated into “reactiontips”. Images were captured over one hour at two-minute intervals. Imageanalysis was used to measure the movement of the interface between redcells and plasma. FIG. 122 shows results as distance of the interfacefrom the plasma meniscus.

The precision of the measurement may be estimated by fitting the data toa polynomial function and calculating the standard deviation of thedifference between the data and the fitted curve (for all samples). Inthe example, this was determined to be 0.038 mm or <2% CV when relatedto the distance moved over one hour. Accordingly, ESR can be measuredprecisely by this method. Another method for determination of ESR is tomeasure the maximum slope of the distance versus time relationship.

Assay Preparation

In some embodiments, tips can be designed to accommodate a plurality ofreactions or assays. Simultaneous measurement of several different assaymixtures and one or more controls or one or more calibrators can be madewithin one tip of the present invention. In doing this we exploit theability to sample several liquid sources by sequential aspiration ofliquids into the same tip. Effective segmentation and separation of theliquids is greatly improved by aspirating in sequence a small volume ofair and a small volume of a wash solution which cleans the surface ofthe tips prior to aspiration of the next liquid of interest.

As described above, tips can have conical shapes. In some embodiments,an assay can require oxygen as a reactant. In such reactions, increasingavailability of oxygen within a reaction can be achieved by moving thesample and/or assay mixture to a wide part of tip to increase surfacearea to volume ratio.

In FIG. 93 and FIG. 94, solutions of bromphenol blue were aspirated intotips. The uppermost segments (aspirated first) 6 uL are from a two-folddilution series (highest concentration (0.0781 mg/mL) to the right ofthe image, with the exception of the left-most tip which is a waterblank). Then air (2 uL), wash solution (2 uL) respectively wereaspirated followed by a 6 uL volume of a fixed concentration controlsolution (0.625 mg/mL).

Using this approach several alternative assay configurations can beachieved, for example:

1. Simultaneous measurement of reagent and/or sample blank and assay

2. Simultaneous measurement of sample, blank and control

3. Within-tip calibration of assay

The table below illustrates some “multiplex types” in which preferredcombinations of assays, controls and calibrators are assembled within atip.

Zone # 1 11 Multiplex Type Top 2 3 4 5 6 7 8 9 10 Bottom Assay withcontrols Air Control1 Air Wash Air Assay Air Wash Control2 Three assaysAir Assay1 Air Wash Air Assay2 Air Wash Air Assay3 Air Calibrationseries Air Cal1 Air Wash Air Cal2 Air Wash Air Cal3 Air Assay with blankAir Assay Air Wash Air Blank Air

Case 2 is shown in FIG. 95.

Serial measurements of blank solutions, sample, controls and calibratorscan also be made with single tips. In this scenario, the tip is filledwith the first solution, read then emptied. The tip can then bere-filled and read with other samples etc. in sequence. The impact of“carry-over” of colored product from one sample to the next is minimizedby either or both:

1. Reading the liquid column in the middle portion well away from thatpart that first comes into contact with the preceding sample

2. Washing the tip between samples.

In order to measure the extent of ‘carry-over” from one liquid segmentto the next, the following procedure was be performed. A small amount(e.g. 6 uL) of a high concentration of bromphenol blue (e.g. 0.625mg/mL) was aspirated into tips, followed by 2 uL of air and 2 uL of washsolution. Finally 6 uL of serial two-fold dilutions of bromphenol blueis aspirated with the following results (highest concentration (0.0781mg/mL) to the right; left most tip is a water blank).

As can be seen from the images shown in FIG. 96 and the 3-color analysisshown in FIG. 97, measurable amounts of the high concentration solutionis transferred into the wash solution.

Average carry-over (from high concentration control to the water wash)is calculated at 1.4%. Since, in effect, the leading zone (proximal tothe earlier slug) of a later slug of liquid acts as second wash step andthe color reading is taken at a location remote from this leading zone(typically at a central zone of the slug), the effective carryover fromone slug to the next is typically much less than 1% and thereforegenerally insignificant. When the dilution series is measured using onlydilution series samples to fill the tips, results are identical withthose obtained above. The above represents a “stress test” designed toevaluate the extent of carry-over.

FIG. 98 shows a tip containing reaction products for two commerciallyavailable assays for ionized calcium, Ca2+ (upper segment) and magnesiumMg2+ (lower segment) that were aspirated into tips for measurement. Ca2+concentrations used in this experiment are 0, 2.5, 5, 10, 20, and 40mg/dL; Mg2+ concentrations are 0, 1.25, 2.5, 5, 10, 20 mg/dL. Assayreaction mixtures (6 uL [Ca2+] and 4 uL [Mg2+]) are well separated using2 uL of air, 3 uL of wash and a further 4 uL of air. Results for eachassay read in this way are essentially identical to those measuredhaving only one assay reaction mixture per tip.

As noted above, the present invention allows for simultaneous evaluationof a plurality of assays. Images can be made of many assay cuvettes inthe same field of view. Specifically, simultaneous evaluation of assaysand controls in the same assay cuvette can be performed. Simultaneousevaluation of several assays in the same assay cuvette can be alsoperformed.

Reaction Environment

A system can comprise a heating block for heating the assay or assayunit and/or for control of the assay temperature. Heat can be used inthe incubation step of an assay reaction to promote the reaction andshorten the duration necessary for the incubation step. A system cancomprise a heating block configured to receive an assay unit of theinvention. The heating block can be configured to receive a plurality ofassay units from a device of the invention. For example, if 8 assays aredesired to be run on a device, the heating block can be configured toreceive 8 assay units. In some embodiments, assay units can be movedinto thermal contact with a heating block using the means for moving theassay units. The heating can be performed by a heating means known inthe art.

Protocol Optimization

Assay protocols for analyzing samples can be optimized in a variety ofmanners. When multiple assays are to be run on a sample, all protocolscan be optimized to the most stringent reaction conditions, or eachassay protocol can be optimized based on the desired performance of aparticular assay.

In some embodiments, a single protocol that can be designed to meet thetest requirements under all possible use cases. For example, on amultiplex cartridge, a single protocol may be specified based on thecase when all tests on the cartridge are to be performed (i.e., thelimiting case). This protocol can be designed to meet the minimal testrequirements, such as the precision and dynamic range for each test onthe cartridge. However, this approach can be suboptimal for alternateuse cases, for example, when only a subset of tests on the cartridge isto be performed. In these cases, by using more sample, some assays canachieve improved performance in terms of sensitivity and precision.There can be a trade-off between how much sample is allocated to anassay and assay sensitivity. For example, an assay which has asensitivity of 1 unit/mL when the sample is diluted 1:100 may be able todetect 0.1 unit/mL if the dilution factor is increased to 1:10. Onedownside of using a lower dilution factor in a multiplexed assay systemwith restricted sample volume can be that the fraction of the samplerequired for this assay is increased by 10-fold even when using theminimal volume to perform the assay. Likewise, assay precision may beimproved by using a higher sample concentration. For example, an assaywhich results in a signal of (say) 0.1 absorbance+/−0.02 (20% signalimprecision) at its limit of detection can be improved by use of 10times the sample concentration such that the signal produced is 10 timesgreater giving a signal of 0.1+/1 0.02 OD at an analyte concentrationten times lower and at signal of 1.0+/−0.02 the imprecision is now only2%. The reason this is the case is that typically assay signal (at thelower range of analyte concentrations) is directly proportional to theanalyte concentration (and therefore to the sample concentration)whereas the signal imprecision can be typically related to the squareroot of the signal and so increases as the square root of analyteconcentration (and sample concentration). Thus, the coefficient ofvariation (CV) of the signal can be inversely proportional to the squareroot of the signal; such that a 10-fold increase in signal correspondsto approximately three-fold decrease in signal CV. Since concentrationCV is typically directly related to signal CV, the concentration CV willdecrease with increased sample concentration (decreased dilution).

Protocols can be optimized to specific use cases rather than the typicalone-size fits all approach described above. For example, the protocolmay be optimized to enhance the precision of each test being performedin the multiplex device. Moreover, some tests may be prioritizedrelative to other tests for optimization. Protocol optimization can bepre-computed for use cases that are known a priori. Protocoloptimization can also be performed in real-time for new use cases notknown a priori. System validation can be performed to span the suite ofuse cases.

One example of protocol optimization is described below comparing twouses cases. For both use cases, 8 uL of undiluted sample is available torun the required tests. In this example, the multiplex cartridge has 20tests on board, where 5 of the tests require 1:5 dilution and 15 of thetests require 1:25 dilution.

In the first use case, all tests are required to be run on the sample.The protocol in this use case (Use-case B) is as follows:

1) Prepare 1:5 dilution (8 uL sample+32 uL diluent)

2) Prepare 1:25 dilution (15 uL 1:5 sample+60 uL diluent)

3) For each test (n=20), mix 5 uL of appropriately diluted sample with10 uL of the reagent. This protocol results in concentration imprecisionof 10% CV for all 20 tests, meeting the minimal requirements. The sampleusage is 1 uL for each 1:5 dilution assay and 0.2 uL for each 1:25dilution assay (for a total of 5*1+15*0.2=8 uL, using all the availablesample).

In the second use case (Use-case “B”) with the same cartridge type, only10 tests are required to be run for the sample, not all 20. Moreover,all these 10 tests would be performed at the 1:25 dilution level inuse-case A. The protocol is optimized for this use case to maximizeprecision for all the tests by using a lower dilution (1:5). Theoptimized protocol for this specific use case is as follows:

1) Prepare 1:5 dilution (8 uL sample+32 uL diluent)

2) For each test (n=10), mix 4 uL of diluted sample with 11 uL ofreagent

Sample usage is 0.8 uL undiluted sample per assay for a total of 8 uL.Since the sample concentration in the assay is increased by 5-foldrelative to that for use-case A, the assay sensitivity is improved by afactor of 5 and the assay imprecision is reduced by about 2.4(5{circumflex over ( )}0.5) fold to about 4.5%.

By re-optimizing the protocol, in use case B employs 5-times as muchoriginal sample for each test, thereby improving overall performance.Note that the above discussion does not account for any imprecision dueto errors in metering of volumes but only addresses errors due toimprecision in measurement of optical signal. Use-case B would have alower imprecision due to imprecision in volumes since it uses fewerpipetting steps. For example if the volume imprecision introduces 5%imprecision in the reported analyte concentration in both use casesthere would be a total analyte imprecision of 11.2% (10{circumflex over( )}2+5{circumflex over ( )}2){circumflex over ( )}0.5 in use-case Acompared with 6.5% (4.5{circumflex over ( )}2+5{circumflex over( )}2){circumflex over ( )}0.5 in use-case B (assuming, as is generallytrue, that factors causing imprecision in assays aggregate as the squareroot of the sum of squares of each source of imprecision).

The effects illustrated above can more easily be seen in the case ofluminescence-based assays where the assay signal is expressed as anumber of photons emitted per unit time. As is the case for counting ofradioactive emissions in for example radioimmunoassay, the signalimprecision is equal to the square root of the signal and thus thesignal CV is 100/(square root of signal). For example, a signal of10,000 counts will have a CV of 1%. In many assays which produce photons(for example chemiluminescence immunoassays, the signal is almostexactly proportional to analyte concentration, at least at the lowerconcentration range). Thus the measured analyte imprecision scales with1/(square root of signal) for concentrations significantly above thelimit of detection. In assays which utilize dilution of the sample, themeasured analyte imprecision will therefore scale as 1/(sampledilution). For example, an assay using a 1:100 dilution of sample willhave signal and concentration CVs about 3.2 fold (10{circumflex over( )}0.5) higher than an assay using a dilution 1:10 (and will also havea sensitivity about 10-times higher).

Reaction Chemistries

A variety of assays may be performed on a fluidic device according tothe present invention to detect an analyte of interest in a sample.Where a label is utilized in the assay, one may choose from a widediversity of labels is available in the art that can be employed forconducting the subject assays. In some embodiments labels are detectableby spectroscopic, photochemical, biochemical, electrochemical,immunochemical, or other chemical means. For example, useful nucleicacid labels include the, fluorescent dyes, electron-dense reagents, andenzymes. A wide variety of labels suitable for labeling biologicalcomponents are known and are reported extensively in both the scientificand patent literature, and are generally applicable to the presentinvention for the labeling of biological components. Suitable labelsinclude, enzymes, fluorescent moieties, chemiluminescent moieties,bioluminescent labels, or colored labels. Reagents defining assayspecificity optionally include, for example, monoclonal antibodies,polyclonal antibodies, aptamers, proteins, nucleic acid probes or otherpolymers such as affinity matrices, carbohydrates or lipids. Detectioncan proceed by any of a variety of known methods, includingspectrophotometric or optical tracking of fluorescent, or luminescentmarkers, or other methods which track a molecule based upon size, chargeor affinity. A detectable moiety can be of any material having adetectable physical or chemical property. Such detectable labels havebeen well-developed in the field of gel electrophoresis, columnchromatography, solid substrates, spectroscopic techniques, and thelike, and in general, labels useful in such methods can be applied tothe present invention. Thus, a label includes without limitation anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, nucleic acid probe-based, electrical, optical thermal,or other chemical means.

In some embodiments the label (such as a colored compound, fluor orenzyme) is coupled directly or indirectly to a molecule to be detected,according to methods well known in the art. In other embodiments, thelabel is attached to a receptor for the analyte (such as an antibody,nucleic acid probe, aptamer etc.). As indicated above, a wide variety oflabels are used, with the choice of label depending on the sensitivityrequired, ease of conjugation of the compound, stability requirements,available instrumentation, and disposal provisions. Non-radioactivelabels are often attached by indirect means. Generally, a receptorspecific to the analyte is linked to a signal-generating moiety.Sometimes the analyte receptor is linked to an adaptor molecule (such asbiotin or avidin) and the assay reagent set includes a binding moiety(such as a biotinylated reagent or avidin) that binds to the adaptor andto the analyte. The analyte binds to a specific receptor on the reactionsite. A labeled reagent can form a sandwich complex in which the analyteis in the center. The reagent can also compete with the analyte forreceptors on the reaction site or bind to vacant receptors on thereaction site not occupied by analyte. The label is either inherentlydetectable or bound to a signal system, such as a detectable enzyme, afluorescent compound, a chemiluminescent compound, or a chemiluminogenicentity such as an enzyme with a luminogenic substrate. A number ofligands and anti-ligands can be used. Where a ligand has a naturalanti-ligand, for example, biotin, thyroxine, digoxigenin, and cortisol,it can be used in conjunction with labeled, anti-ligands. Alternatively,any haptenic or antigenic compound can be used in combination with anantibody.

Enzymes of interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl groups, andumbelliferone. Chemiluminescent compounds include dioxetanes, acridiniumesters, luciferin, and 2,3-dihydrophthalazinediones, such as luminol.

Methods of detecting labels are well known to those of skilled in theart. Thus, for example, where the label is fluorescent, it may bedetected by exciting the fluorochrome with light of an appropriatewavelength and detecting the resulting fluorescence by, for example,microscopy, visual inspection, via photographic film, by the use ofelectronic detectors such as digital cameras, charge coupled devices(CCDs) or photomultipliers and phototubes, or other detection devices.Similarly, enzymatic labels are detected by providing appropriatesubstrates for the enzyme and detecting the resulting reaction productspectroscopically or by digital imaging (the subject of the presentinvention). Finally, simple colorimetric labels are often detectedsimply by observing the color associated with the label. For example,colloidal gold sols often appear pink, while various beads doped withdyes are strongly colored.

In some embodiments the detectable signal may be provided byluminescence sources Luminescence is the term commonly used to refer tothe emission of light from a substance for any reason other than a risein its temperature. In general, atoms or molecules emit photons ofelectromagnetic energy (e.g., light) when they transition from anexcited state to a lower energy state (usually the ground state). If theexciting agent is a photon, the luminescence process is referred to asphotoluminescence or fluorescence. If the exciting cause is an electron,the luminescence process can be referred to as electroluminescence. Morespecifically, electroluminescence results from the direct injection andremoval of electrons to form an electron-hole pair, and subsequentrecombination of the electron-hole pair to emit a photon Luminescencewhich results from a chemical reaction is usually referred to aschemiluminescence. Luminescence produced by a living organism is usuallyreferred to as bioluminescence. If photoluminescence is the result of aspin allowed transition (e.g., a single-singlet transition,triplet-triplet transition), the photoluminescence process is usuallyreferred to as fluorescence. Typically, fluorescence emissions do notpersist after the exciting cause is removed as a result of short-livedexcited states which may rapidly relax through such spin allowedtransitions. If photoluminescence is the result of a spin forbiddentransition (e.g., a triplet-singlet transition), the photoluminescenceprocess is usually referred to as phosphorescence. Typically,phosphorescence emissions persist long after the exciting cause isremoved as a result of long-lived excited states which may relax onlythrough such spin-forbidden transitions. A luminescent label may haveany one of the above-described properties.

Suitable chemiluminescent sources include a compound which becomeselectronically excited by a chemical reaction and may then emit lightwhich serves as the detectible signal or donates energy to a fluorescentacceptor. A diverse number of families of compounds have been found toprovide chemiluminescence under a variety of conditions. One family ofcompounds is 2,3-dihydro-1,4-phthalazinedione. A frequently usedcompound is luminol, which is a 5-amino compound. Other members of thefamily include the 5-amino-6, 7, 8-trimethoxy- and thedimethylamino[ca]benz analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino and -methoxy substituents.Chemiluminescence may also be obtained with oxalates, usually oxalylactive esters, for example, p-nitrophenyl and a peroxide such ashydrogen peroxide, under basic conditions. Other useful chemiluminescentcompounds that are also known include —N-alkyl acridinum esters anddioxetanes. Alternatively, luciferins may be used in conjunction withluciferase or lucigenins to provide bioluminescence. Especiallypreferred chemiluminescent sources are “luminogenic” enzyme substratessuch as dioxetane-phosphate esters. These are not luminescent butproduce luminescent products when acted on by phosphatases such asalkaline phosphatase. The use of luminogenic substrates for enzymes isparticularly preferred because the enzyme acts as an amplifier capableof converting thousands of substrate molecules per second to product.Luminescence methods are also preferred because the signal (light) canbe detected both very sensitively and over a huge dynamic range usingPMTs.

The term analytes as used herein includes without limitation drugs,prodrugs, pharmaceutical agents, drug metabolites, biomarkers such asexpressed proteins and cell markers, antibodies, serum proteins,cholesterol and other metabolites, electrolytes, metal ions,polysaccharides, nucleic acids, biological analytes, biomarkers, genes,proteins, hormones, or any combination thereof. Analytes can becombinations of polypeptides, glycoproteins, polysaccharides, lipids,and nucleic acids.

The system can be used to detect and/or quantify a variety of analytes.For example, analytes that can be detected and/or quantified includeAlbumin, Alkaline Phosphatase, ALT, AST, Bilirubin (Direct), Bilirubin(Total), Blood Urea Nitrogen (BUN), Calcium, Chloride, Cholesterol,Carbon Dioxide (CO₂), Creatinine, Gamma-glutamyl-transpeptidase (GGT),Globulin, Glucose, HDL-cholesterol, Hemoglobin, Homocysteine, Iron,Lactate Dehydrogenase, Magnesium, Phosphorous, Potassium, Sodium, TotalProtein, Triglycerides, and Uric Acid. The detection and/orquantification of these analytes can be performed using optical,electrical, or any other type of measurements.

Of particular interest are biomarkers which are associated with aparticular disease or with a specific disease stage. Such analytesinclude but are not limited to those associated with autoimmunediseases, obesity, hypertension, diabetes, neuronal and/or musculardegenerative diseases, cardiac diseases, endocrine disorders, metabolicdisorders, inflammation, cardiovascular diseases, sepsis, angiogenesis,cancers, Alzheimer's disease, athletic complications, and anycombinations thereof.

Of also interest are biomarkers that are present in varying abundance inone or more of the body tissues including heart, liver, prostate, lung,kidney, bone marrow, blood, skin, bladder, brain, muscles, nerves, andselected tissues that are affected by various disease, such as differenttypes of cancer (malignant or non-metastatic), autoimmune diseases,inflammatory or degenerative diseases.

Also of interest are analytes that are indicative of a microorganism,virus, or Chlamydiaceae. Exemplary microorganisms include but are notlimited to bacteria, viruses, fungi and protozoa. Analytes that can bedetected by the subject method also include blood-born pathogensselected from a non-limiting group that consists of Staphylococcusepidermidis, Escherichia coli, methicillin-resistant Staphylococcusaureus (MSRA), Staphylococcus aureus, Staphylococcus hominis,Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus capitis,Staphylococcus warneri, Klebsiella pneumoniae, Haemophilus influenzae,Staphylococcus simulans, Streptococcus pneumoniae and Candida albicans.

Analytes that can be detected by the subject method also encompass avariety of sexually transmitted diseases selected from the following:gonorrhea (Neisseria gonorrhoeae), syphilis (Treponena pallidum),clamydia (Clamyda tracomitis), nongonococcal urethritis (Ureaplasmurealyticum), yeast infection (Candida albicans), chancroid (Haemophilusducreyi), trichomoniasis (Trichomonas vaginalis), genital herpes (HSVtype I & II), HIV I, HIV II and hepatitis A, B, C, G, as well ashepatitis caused by TTV.

Additional analytes that can be detected by the subject methodsencompass a diversity of respiratory pathogens including but not limitedto Pseudomonas aeruginosa, methicillin-resistant Staphlococccus aureus(MSRA), Klebsiella pneumoniae, Haemophilis influenzae, Staphylococcusaureus, Stenotrophomonas maltophilia, Haemophilis parainfluenzae,Escherichia coli, Enterococcus faecalis, Serratia marcescens,Haemophilis parahaemolyticus, Enterococcus cloacae, Candida albicans,Moraxiella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii,Enterococcus faecium, Klebsella oxytoca, Pseudomonas fluorscens,Neiseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii,Klebsella pneumoniae Legionella pneumophila, Mycoplasma pneumoniae, andMycobacterium tuberculosis.

Listed below are additional exemplary markers according to the presentinvention: Theophylline, CRP, CKMB, PSA, Myoglobin, CA125, Progesterone,TxB2, 6-keto-PGF-1-alpha, and Theophylline, Estradiol, Lutenizinghormone, Triglycerides, Tryptase, Low density lipoprotein Cholesterol,High density lipoprotein Cholesterol, Cholesterol, IGFR.

Exemplary liver markers include without limitation LDH, (LDS),Alanine-aminotransferase (ALT), Arginase 1 (liver type),Alpha-fetoprotein (AFP), Alkaline phosphatase, Lactate dehydrogenase,and Bilirubin.

Exemplary kidney markers include without limitation TNFa Receptor,Cystatin C, Lipocalin-type urinary prostaglandin D, synthatase (LPGDS),Hepatocyte growth factor receptor, Polycystin 2, Polycystin 1,Fibrocystin, Uromodulin, Alanine, aminopeptidase,N-acetyl-B-D-glucosaminidase, Albumin, and Retinol-binding protein(RBP).

Exemplary heart markers include without limitation Troponin I (TnI),Troponin T (TnT), Creatine dinase (CK), CKMB, Myoglobin, Fatty acidbinding protein (FABP), C-reactive protein (CRP), Fibrinogen D-dimer,S-100 protein, Brain natriuretic peptide (BNP), NT-proBNP, PAPP-A,Myeloperoxidase (MPO), Glycogen phosphorylase isoenzyme BB (GPBB),Thrombin Activatable Fibrinolysis Inhibitor (TAFI), Fibrinogen, Ischemiamodified albumin (IMA), Cardiotrophin-1, and MLC-I (Myosin LightChain-I).

Exemplary pancrease markers include without limitation Amylase,Pancreatitis-Associated protein (PAP-1), and Regeneratein proteins(REG).

Exemplary muscle tissue markers include without limitation Myostatin.

Exemplary blood markers include without limitation Erythopoeitin (EPO).

Exemplary bone markers include without limitation, Cross-linkedN-telopeptides of bone type I collagen (NTx), Carboxyterminalcross-linking telopeptide of bone collagen, Lysyl-pyridinoline(deoxypyridinoline), Pyridinoline, Tartrate-resistant acid phosphatase,Procollagen type I C propeptide, Procollagen type I N propeptide,Osteocalcin (bone gla-protein), Alkaline phosphatase, Cathepsin K, COMP(Cartillage Oligimeric Matrix Protein), Osteocrin, Osteoprotegerin(OPG), RANKL, sRANK, TRAP 5 (TRACP 5), Osteoblast Specific Factor 1(OSF-1, Pleiotrophin), Soluble cell adhesion molecules, sTfR, sCD4,sCD8, sCD44, and Osteoblast Specific Factor 2 (OSF-2, Periostin).

In some embodiments markers according to the present invention aredisease specific. Exemplary cancer markers include without limitationPSA (total prostate specific antigen), Creatinine, Prostatic acidphosphatase, PSA complexes, Prostrate-specific gene-1, CA 12-5,Carcinoembryonic Antigen (CEA), Alpha feto protein (AFP), hCG (Humanchorionic gonadotropin), Inhibin, CAA Ovarian C1824, CA 27.29, CA 15-3,CAA Breast C1924, Her-2, Pancreatic, CA 19-9, CAA pancreatic,Neuron-specific enolase, Angiostatin DcR3 (Soluble decoy receptor 3),Endostatin, Ep-CAM (MK-1), Free Immunoglobulin Light Chain Kappa, FreeImmunoglobulin Light Chain Lambda, Herstatin, Chromogranin A,Adrenomedullin, Integrin, Epidermal growth factor receptor, Epidermalgrowth factor receptor-Tyrosine kinase, Pro-adrenomedullin N-terminal 20peptide, Vascular endothelial growth factor, Vascular endothelial growthfactor receptor, Stem cell factor receptor, c-kit/KDR, KDR, and Midkine.

Exemplary infectious disease conditions include without limitation:Viremia, Bacteremia, Sepsis, and markers: PMN Elastase, PMNelastase/α1-PI complex, Surfactant Protein D (SP-D), HBVc antigen, HBVsantigen, Anti-HBVc, Anti-HIV, T-supressor cell antigen, T-cell antigenratio, T-helper cell antigen, Anti-HCV, Pyrogens, p24 antigen,Muramyl-dipeptide.

Exemplary diabetes markers include without limitation C-Peptide,Hemoglobin A1c, Glycated albumin, Advanced glycosylation end products(AGEs), 1,5-anhydroglucitol, Gastric Inhibitory Polypeptide, Glucose,Hemoglobin A1c, ANGPTL3 and 4.

Exemplary inflammation markers include without limitation Rheumatoidfactor (RF), Antinuclear Antibody (ANA), C-reactive protein (CRP), ClaraCell Protein (Uteroglobin).

Exemplary allergy markers include without limitation Total IgE andSpecific IgE.

Exemplary autism markers include without limitation Ceruloplasmin,Metalothioneine, Zinc, Copper, B6, B12, Glutathione, Alkalinephosphatase, and Activation of apo-alkaline phosphatase.

Exemplary coagulation disorders markers include without limitationb-Thromboglobulin, Platelet factor 4, Von Willebrand factor.

In some embodiments a marker may be therapy specific. Markers indicativeof the action of COX inhibitors include without limitation TxB2 (Cox-1),6-keto-PGF-1-alpha (Cox 2), 11-Dehydro-TxB-1a (Cox-1).

Other markers of the present invention include without limitationLeptin, Leptin receptor, and Procalcitonin, Brain S100 protein,Substance P, 8-Iso-PGF-2a.

Exemplary geriatric markers include without limitation, Neuron-specificenolase, GFAP, and S100B.

Exemplary markers of nutritional status include without limitationPrealbumin, Albumin, Retinol-binding protein (RBP), Transferrin,Acylation-Stimulating Protein (ASP), Adiponectin, Agouti-Related Protein(AgRP), Angiopoietin-like Protein 4 (ANGPTL4, FIAF), C-peptide, AFABP(Adipocyte Fatty Acid Binding Protein, FABP4), Acylation-StimulatingProtein (ASP), EFABP (Epidermal Fatty Acid Binding Protein, FABP5),Glicentin, Glucagon, Glucagon-Like Peptide-1, Glucagon-Like Peptide-2,Ghrelin, Insulin, Leptin, Leptin Receptor, PYY, RELMs, Resistin, amdsTfR (soluble Transferrin Receptor).

Exemplary markers of Lipid metabolism include without limitationApo-lipoproteins (several), Apo-A1, Apo-B, Apo-C-CII, Apo-D, Apo-E.

Exemplary coagulation status markers include without limitation FactorI: Fibrinogen, Factor II: Prothrombin, Factor III: Tissue factor, FactorIV: Calcium, Factor V: Proaccelerin, Factor VI, Factor VII:Proconvertin, Factor VIII: Anti-hemolytic factor, Factor IX: Christmasfactor, Factor X: Stuart-Prower factor, Factor XI: Plasma thromboplastinantecedent, Factor XII: Hageman factor, Factor XIII: Fibrin-stabilizingfactor, Prekallikrein, High-molecular-weight kininogen, Protein C,Protein S, D-dimer, Tissue plasminogen activator, Plasminogen,a2-Antiplasmin, Plasminogen activator inhibitor 1 (PAI1).

Exemplary monoclonal antibodies include those for EGFR, ErbB2, andIGF1R.

Exemplary tyrosine kinase inhibitors include without limitation Abl,Kit, PDGFR, Src, ErbB2, ErbB 4, EGFR, EphB, VEGFR1-4, PDGFRb, FLt3,FGFR, PKC, Met, Tie2, RAF, and TrkA.

Exemplary Serine/Threonine Kinase Inhibitors include without limitationAKT, Aurora A/B/B, CDK, CDK (pan), CDK1-2, VEGFR2, PDGFRb, CDK4/6,MEK1-2, mTOR, and PKC-beta.

GPCR targets include without limitation Histamine Receptors, SerotoninReceptors, Angiotensin Receptors, Adrenoreceptors, MuscarinicAcetylcholine Receptors, GnRH Receptors, Dopamine Receptors,Prostaglandin Receptors, and ADP Receptors.

Cholesterol

Measurement of metabolites can be performed by production of a coloredproduct using oxidases (such as cholesterol oxidase) (to make H2O2) andhorse-radish peroxidase plus a chromogen (such asN-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, sodium salt[“DAOS” plus amino anti-pyrene] to form a colored product such as aTrinder dye). One example of such chemistry is shown in FIG. 52 and FIG.53.

NADH or NADPH

Production or consumption of NADH or NADPH are frequently used inclinical assays. This is because these coenzymes are common substratesfor enzymes. For example, measurement of enzymes of clinical interestsuch as lactate dehydogenase (LDH) can be measured by the rate ofproduction of NADH. Since NADH absorbs light maximally at 340 nm and (1)polystyrene and other plastics transmit light poorly in the near UV, (2)White light sources produce little light in the near UV and (3) cameraand scanner sensors have low sensitivity to near UV light, it is notpractical to measure NADH by three color image analysis. To deal withthis issue NADH can be converted to a colored product using tetrazoliumsalts such as Water Soluble Tetrazolium (e.g. WST-1 (Dojindo MolecularTechnologies) plus an “electron mediator” such as 1-Methoxyphenazinemethosulfate (PMS).

In some embodiments, assays that produce or consume NADH or NADPH can bepaired with other reactions that allow for colorimetric measurement. Forexample, NADH or NADPH can be used to reduce compounds such as2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt (WST-1) to a colored formezan dye as shown below withthe use of phenazine methosulfate as an electron mediator, as shown inFIG. 54.

As shown in FIG. 73, when NADH, WST-1 and PMS are combined at millimolarconcentrations, a yellow product (shown in tips indicated as Mixture) isformed.

Using this chemistry, an assay for LDH was set up. Lactate (mM), NAD(mM) and LDH were combined and incubated at 37° C. for 10 minutes beforeaddition of WST-1 and PMS. A good dose-response to LDH was obtained asshown in FIG. 74 for two-fold serial dilutions of LDH (1000 IU/L) (leftto right) corresponding to the OD 450 nm values shown in the graph inFIG. 75.

Alkaline Phosphatase

In other embodiments, assays utilizing enzymes such as alkalinephosphatase can be measured using a chromogenic substrate such asp-nitrophenyl phosphate. The enzymatic reaction can make p-nitrophenolwhich is yellow in alkaline conditions.

Metal Ions

Measurements can also be performed on assays that form colored complex,such as between a metal ion a chelating dye which changes color onbinding. For example, o-Cresolphthalein Complexone (shown in FIG. 55)forms a complex with calcium, which has a different color than thereagent. The general scheme of such assays is: Chelating dye (color1)+M^(N+) <-> Chelating dye: M^(N+):(Color 2)

Optical signals can also be measured for metal ion assays usingmetal-dependent enzymes. For example, sodium ions can be determinedenzymatically via sodium dependent β-galactosidase activity witho-nitro-phenyl galactoside (ONPG) as the substrate. The absorbance at405 nm of the product o-nitrophenol is proportional to the sodiumconcentration.

ELISAs

Assays can be performed for analytes by color-forming ELISAs. Many ELISAmethods are known which generate color using enzymes such as horseradishperoxidase, alkaline phosphatase and β-galactosidase with chromogenicsubstrates such as o-phenylene diamine, p-nitrophenyl phosphate, ando-nitrophenyl galactoside respectively. Such assays can be readilyperformed and read by the subject invention.

Luminogenic Immunoassays

Luminogenic immunoassays can also be performed. Assays can utilizechemiluminogenic entities such as an enzyme with a luminogenicsubstrate. For example, chemiluminescent compounds include dioxetanes,acridinium esters, luciferin, and 2,3-dihydrophthalazinediones, such asluminol.

Furthermore, suitable chemiluminescent sources include a compound whichbecomes electronically excited by a chemical reaction and may then emitlight which serves as the detectible signal or donates energy to afluorescent acceptor. A diverse number of families of compounds havebeen found to provide chemiluminescence under a variety of conditions.One family of compounds is 2,3-dihydro-1,4-phthalazinedione. Afrequently used compound is luminol, which is a 5-amino compound. Othermembers of the family include the 5-amino-6, 7, 8-trimethoxy- and thedimethylamino[ca]benz analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino and -methoxy substituents.Chemiluminescence may also be obtained with oxalates, usually oxalylactive esters, for example, p-nitrophenyl and a peroxide such ashydrogen peroxide, under basic conditions. Other useful chemiluminescentcompounds that are also known include N-alkyl acridinum esters anddioxetanes. Alternatively, luciferins may be used in conjunction withluciferase or lucigenins to provide bioluminescence.

Nucleic Acid Amplification

Assays that can be performed also include nucleic acid amplification.Among these assays, isothermal amplification and Loop-MediatedIsothermal Amplification Assays (LAMP) are examples. Nucleic acidamplification can be used to produce visibly turbid, fluorescent orcolored assay reaction products for analytes such as nucleic acidtargets (genes etc.). Nucleic acid amplification technology can be usedfor isothermal amplification of specific DNA and RNA targets. Additionalinformation on isothermal nucleic acid amplification is described inGoto et al., “Colorimetric detection of loop-mediated isothermalamplification reaction by using hydroxy naphthol blue”, BioTechniques,Vol. 46, No. 3, March 2009, 167-172.

Nucleic acid amplification can be used to measure DNA and, coupled withthe use of reverse transcriptase, RNA. Once the reaction has occurred,the amplified product can be detected optically using intercalating dyesor chromogenic reagents that react with released pyrophosphate generatedas a side product of the amplification.

The reaction can be visualized by changes (increases) in color,fluorescence or turbidity. Very small copy numbers of DNA can bedetected in less than one hour. This technology can advantageously beread out in the present invention using three-color image analysis. Asshown below, images of isothermal nucleic acid amplification assayreaction products can be measured by (1) back lit-illumination(transmission optics) measuring absorbance of light, (2) images capturedby a digital camera of light transmitted through a reaction product or(3) fluorescent light images generated by illumination of reactionproducts with a UV source (or any other appropriate light source)captured by a digital camera.

The nucleic acid amplification assay is generally performed in a“one-pot” format where sample and reagents are combined in a sealed tubeand incubated at elevated temperature. In some formats, the reaction canbe monitored in real time by changes in optical properties. In otherassay formats the reaction is stopped and reaction products visualizedafter adding a chromogenic or fluorogenic reagent. The present inventionallows for the reading of nucleic acid amplification assay productsdirectly in the reaction vessel or after aspiration into the tipsdescribed herein.

Turbidity

The invention also provides for optical turbidimetric assays. Forexample, immunoassays can be set up by measurement of the agglutinationof small latex particles (50-300 nm). In these assays the particles canbe coated with an antigen and/or antibody and agglutination occurs whena binding counterpart in the sample such as antibody or antigen isadded. Assays can be set up as direct (e.g. antibody on the particlereacting with a multi-epitope protein or biomarker) or the competitivemode (e.g. drug hapten on particle reacts with anti-drug antibody incompetition with free drug in the sample). The dispersion of latexbecomes more turbid and the turbidity can be measured as decreasedtransmission of light using 3-color optics.

Similarly, assays based on the agglutination of large latex particles(diameter about 1 um) or red blood cells can be measured. Assayconfiguration is similar to turbidimetric assays as disclosed above, butthe measurement can be by image analysis (scanner or camera measurement)using software to interpret the number and size of the agglutinates.

Reagents for performing reaction chemistries can be included in thecartridges described here, such as in pipette tips. The reagents can bestored as liquids or in dried, lyophilized, or glassy forms.

Localized Reagents

In some embodiments, the location and configuration of a reaction siteis an important element in an assay device. Most, if not all, disposableimmunoassay devices have been configured with their capture surface asan integral part of the device.

In one embodiment, a molded plastic assay unit is either commerciallyavailable or can be made by injection molding with precise shapes andsizes. For example, the characteristic dimension can be a diameter of0.05-3 mm or can be a length of 3 to 30 mm. The units can be coated withcapture reagents using method similar to those used to coat microtiterplates but with the advantage that they can be processed in bulk byplacing them in a large vessel, adding coating reagents and processingusing sieves, holders, and the like to recover the pieces and wash themas needed.

The assay unit (e.g. encompassing the tip disclosed herein, tips,vessels, or any other containers) can offer a rigid support on which areactant can be immobilized. The assay unit is also chosen to provideappropriate characteristics with respect to interactions with light. Forexample, the assay unit can be made of a material, such asfunctionalized glass, Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon,or any one of a wide variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,polycarbonate, polypropylene, polymethylmethacrylate (PMMA),acrylonitrile-butadiene-styrene (ABS), or combinations thereof. In anembodiment, an assay unit comprises polystyrene. In some embodiments,the assay unit may be formed from a homogeneous material, heterogeneousmaterial, clad material, coated material, impregnated material, and/orembedded material. Other appropriate materials may be used in accordancewith the present invention. A transparent reaction site may beadvantageous. In addition, in the case where there is an opticallytransmissive window permitting light to reach an optical detector, thesurface may be advantageously opaque and/or preferentially lightscattering. In some embodiments, the assay unit may be formed from atransparent material. Alternatively, a portion of the assay unit may beformed from a transparent material.

The assay unit may have a reagent coated thereon and/or impregnatedtherein. In some embodiments, the reagent may be a capture reagentcapable of immobilizing a reactant on a capture surface. The reactantmay be a cell and/or analyte, or any other reactant described elsewhereherein. In some embodiments, the reagent may be a molecule that may be acell capture agent. A cell capture agent may anchor to the surface ofdesired cells during fluid transport. In some embodiments, the capturereagents may be an antibody, peptide, organic molecule (e.g., which mayhave a lipid chain, lipophilic molecule), polymer matrix, protein,protein composite, glycoprotein, that may interact with the cellmembrane. Capture reagents may be molecules, cross-linked molecules,nanoparticles, nanostructures, and/or scaffolds. In some embodiments,microstructures may be provided that may become an analysis mechanism ina vessel. Capture reagents (which may include capture structures formedby the assay unit material) may allow cells to be tethered, bound,and/or trapped.

The capture reagents may immobilize a reactant, such as a cell, duringprocessing. Capture techniques may be chemical, physical, electrical,magnetic, mechanical, size-related, density-related, or any combinationthereof. In some embodiments, the capture reagents may be used toconcentrate reactants, such as cells, at a desired location. Forexample, an assay unit may be coated with the capture reagents, whichmay cause cells to be captured at the assay unit surface, thusconcentrating the cells on the captured surface. The capture reagentsmay keep the captured reactant immobilized on the cell surface. This mayaid in keeping the reactants (e.g., cells, analytes) stationary duringimaging.

Immobilizing the reactants may be useful for applications where theremay be long acquisition times for reactions and/or detection. Forexample, a number of imaging applications may require extended exposuretimes (˜1 min) or imaging of small objects (<1 um) which may havesignificant Brownian motion.

In some embodiments, the capture reagents may be formed from materialsthat may provide little or no background for imaging. In some instances,the material of the assay unit may provide little or no background forimaging. The capture reagents may be selected so that they do notinterfere with, or only have a small interference with, imaging and/ordetection.

A reactant immobilized at the capture surface can be anything useful fordetecting an analyte of interest in a sample of bodily fluid. Forinstance, such reactants include, without limitation, nucleic acidprobes, antibodies, cell membrane receptors, monoclonal antibodies,antisera, and aptamers reactive with a specific analyte. Variouscommercially available reactants such as a host of polyclonal andmonoclonal antibodies specifically developed for specific analytes canbe used.

One skilled in the art will appreciate that there are many ways ofimmobilizing various reactants onto a support where reaction can takeplace. The immobilization may be covalent or noncovalent, via a linkermoiety, or tethering them to an immobilized moiety. Non-limitingexemplary binding moieties for attaching either nucleic acids orproteinaceous molecules such as antibodies to a solid support includestreptavidin or avidin/biotin linkages, carbamate linkages, esterlinkages, amide, thiolester, (N)-functionalized thiourea, functionalizedmaleimide, amino, disulfide, amide, hydrazone linkages, and amongothers. In addition, a silyl moiety can be attached to a nucleic aciddirectly to a substrate such as glass using methods known in the art.Surface immobilization can also be achieved via a Poly-L Lysine tether,which provides a charge-charge coupling to the surface.

The assay units can be dried following the last step of incorporating acapture surface. For example, drying can be performed by passiveexposure to a dry atmosphere or via the use of a vacuum manifold and/orapplication of clean dry air through a manifold or by lyophilization.

A capture surface may be applied to an assay unit using any technique.For example, the capture surface may be painted on, printed on,electrosprayed on, embedded in the material, impregnating the material,or any other technique. The capture reagents may be coated to the assayunit material, incorporated in the material, co-penetrate the material,or may be formed from the material. For example, a reagent, such as acapture reagent may be embedded in a polymer matrix that can be used asa sensor. In some embodiments, one or more small particles, such as ananoparticle, a microparticle, and/or a bead, may be coated and/orimpregnated with reagents. In some embodiments, the capture reagents maybe part of the assay unit material itself, or may be something that isadded to the material.

In many embodiments, an assay unit is designed to enable the unit to bemanufactured in a high volume, rapid manufacturing processes. Forexample, tips can be mounted in large-scale arrays for batch coating ofthe capture surface into or onto the tip. In another example, tips canbe placed into a moving belt or rotating table for serial processing. Inyet another example, a large array of tips can be connected to vacuumand/or pressure manifolds for simple processing.

A capture reagent may be applied to an assay unit during any point inthe process. For example, the capture reagent may be applied to theassay unit during manufacturing. The capture reagent may be applied tothe assay unit prior to shipping the assay unit to a destination.Alternatively, the capture reagent may be applied to the assay unitafter the assay unit has been shipped. In some instances, the capturereagent may be applied to the assay unit at a point of use, such as apoint of service location.

In some embodiments, the capture reagent may cover an entire surface orregion of the assay unit. The capture reagent may be provided on aninner surface of the assay unit. In some embodiments, the capturereagent may cover portions or sections of an assay unit surface. Thecapture reagent may be provided on a surface in a pattern. A unit mayhave portions of the surface that have a capture reagent appliedthereon, and portions of the surface that do not have a capture reagentapplied thereon. For example, there may be coated and non-coatedregions. A capture reagent may be applied in a surface in accordancewith a geometric choice of how the capture reagent is to be applied. Forexample, the capture reagent may be applied in dots, rows, columns,arrays, regions, circles, rings, or any other shape or pattern. Thecapture reagents may be applied at desired positions on the surface.

A plurality of capture reagents may optionally be applied to an assayunit. In some embodiments, the plurality of capture reagents may beapplied so that the different capture reagents do not overlap (e.g., thedifferent capture reagents are not applied to the same region or area).Alternatively, they may overlap (e.g., the different capture reagentsmay be applied to the same region or area). Space without any capturereagents may or may not be provided between regions with differentcapture reagents. The different capture reagents may be used toimmobilize different reactants. For example, different capture reagentsmay be used to immobilize different cells and/or analytes on the capturesurface. By using a plurality of capture reagents patterned in selectedregions, a plurality of reactants may be detected from the same assayunit. In some embodiments, two or more, three or more, four or more,five or more, seven or more, ten or more, fifteen or more, twenty ormore, thirty or more, forty or more, fifty or more, seventy or more, 100or more, 150 or more, 200 or more, or 300 or more different capturereagents may be applied to a surface of an assay unit. The differentcapture reagents may be applied in any pattern or shape. For example,different capture reagents may be applied as an array or series of ringson an inner surface of an assay unit. For example, different capturereagents may be applied on an inner surface of a tip, vessel, container,cuvette, or any other container described elsewhere herein.

The location of the different capture reagents on the assay unit may beknown prior to detection of the captured reactants. In some embodiments,the assay unit may have an identifier that may indicate the type ofassay unit and/or the pattern of capture agents therein. Alternativelythe location of the different capture reagents of the assay unit may notbe known prior to detection of the captured reactants. The location ofthe different capture reagents may be determined based on detectedpatterns of captured reactants.

The capture reagents may be applied using any technique, such as thosedescribed elsewhere herein. In some instances, masking or lithographictechniques may be used to apply different capture reagents.

Any description herein of a capture reagent and/or coating applied to anassay unit may apply to any other units or containers describedelsewhere herein, including but not limited to tips, vessels, cuvettes,or reagent units.

Reagent Assemblies

In many embodiments of the invention the reagent units are modular. Thereagent unit can be designed to enable the unit to be manufactured in ahigh volume, rapid manufacturing processes. For example, many reagentunits can be filled and sealed in a large-scale process simultaneously.The reagent units can be filled according to the type of assay or assaysto be run by the device. For example, if one user desires differentassays than another user, the reagent units can be manufacturedaccordingly to the preference of each user, without the need tomanufacture an entire device. In another example, reagent units can beplaced into a moving belt or rotating table for serial processing.

In another embodiment, the reagent units are accommodated directly intocavities in the housing of a device. In this embodiment, a seal can bemade onto areas of housing surrounding the units.

Reagents according to the present invention include without limitationwash buffers, enzyme substrates, dilution buffers, conjugates,enzyme-labeled conjugates, DNA amplifiers, sample diluents, washsolutions, sample pre-treatment reagents including additives such asdetergents, polymers, chelating agents, albumin-binding reagents, enzymeinhibitors, enzymes, anticoagulants, red-cell agglutinating agents,antibodies, or other materials necessary to run an assay on a device. Anenzyme-labeled conjugate can be either a polyclonal antibody ormonoclonal antibody labeled with an enzyme that can yield a detectablesignal upon reaction with an appropriate substrate. Non-limitingexamples of such enzymes are alkaline phosphatase and horseradishperoxidase. In some embodiments, the reagents comprise immunoassayreagents. In general, reagents, especially those that are relativelyunstable when mixed with liquid, are confined separately in a definedregion (for example, a reagent unit) within the device.

In some embodiments, a reagent unit contains approximately about 5microliters to about 1 milliliter of liquid. In some embodiments, theunit may contain about 20-200 microliters of liquid. In a furtherembodiment, the reagent unit contains 100 microliters of fluid. In anembodiment, a reagent unit contains about 40 microliters of fluid. Thevolume of liquid in a reagent unit may vary depending on the type ofassay being run or the sample of bodily fluid provided. In anembodiment, the volumes of the reagents do not have to predetermined,but must be more than a known minimum. In some embodiments, the reagentsare initially stored dry and dissolved upon initiation of the assaybeing run on the device.

In an embodiment, the reagent units can be filled using a siphon, afunnel, a pipette, a syringe, a needle, or a combination thereof. Thereagent units may be filled with liquid using a fill channel and avacuum draw channel. The reagent units can be filled individually or aspart of a bulk manufacturing process.

In an embodiment, an individual reagent unit comprises a differentreagent as a means of isolating reagents from each other. The reagentunits may also be used to contain a wash solution or a substrate. Inaddition, the reagent units may be used to contain a luminogenicsubstrate. In another embodiment, a plurality of reagents are containedwithin a reagent unit.

In some instances, the setup of the device enables the capability ofpre-calibration of assay units and the reagent units prior to assemblyof disposables of the subject device.

Aptamer Binding Assays

The subject invention enables a variety of assay methods based on theuse of binding elements that specifically bind to one or more analytesin a sample. In general, a binding element is one member of a bindingpair capable of specifically and selectively binding to the other memberof the binding pair in the presence of a plurality of differentmolecules. Examples of binding elements include, but are not limited to,antibodies, antigens, metal-binding ligands, nucleic acid probes andprimers, receptors and reactants as described herein, and aptamers. Insome embodiments, a binding element used to detect an analyte is anaptamer. The term “aptamer” is used to refer to a peptide, nucleic acid,or a combination thereof that is selected for the ability tospecifically bind one or more target analytes. Peptide aptamers areaffinity agents that generally comprise one or more variable loopdomains displayed on the surface of a scaffold protein. A nucleic acidaptamer is a specific binding oligonucleotide, which is anoligonucleotide that is capable of selectively forming a complex with anintended target analyte. The complexation is target-specific in thesense that other materials, such as other analytes that may accompanythe target analyte, do not complex to the aptamer with as great anaffinity. It is recognized that complexation and affinity are a matterof degree; however, in this context, “target-specific” means that theaptamer binds to target with a much higher degree of affinity than itbinds to contaminating materials. The meaning of specificity in thiscontext is thus similar to the meaning of specificity as applied toantibodies, for example. The aptamer may be prepared by any knownmethod, including synthetic, recombinant, and purification methods.Further, the term “aptamer” also includes “secondary aptamers”containing a consensus sequence derived from comparing two or more knownaptamers to a given target.

In general, nucleic acid aptamers are about 9 to about 35 nucleotides inlength. In some embodiments, a nucleic acid aptamer is at least 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 80, 90, 100, or more residues in length. Although theoligonucleotides of the aptamers generally are single-stranded ordouble-stranded, it is contemplated that aptamers may sometimes assumetriple-stranded or quadruple-stranded structures. In some embodiments, anucleic acid aptamer is circular, such as in US20050176940. The specificbinding oligonucleotides of the aptamers should contain thesequence-conferring specificity, but may be extended with flankingregions and otherwise derivatized or modified. The aptamers found tobind to a target analyte may be isolated, sequenced, and thenre-synthesized as conventional DNA or RNA moieties, or may be modifiedoligomers. These modifications include, but are not limited toincorporation of: (1) modified or analogous forms of sugars (e.g. riboseand deoxyribose); (2) alternative linking groups; or (3) analogous formsof purine and pyrimidine bases.

Nucleic acid aptamers can comprise DNA, RNA, functionalized or modifiednucleic acid bases, nucleic acid analogues, modified or alternativebackbone chemistries, or combinations thereof. The oligonucleotides ofthe aptamers may contain the conventional bases adenine, guanine,cytosine, and thymine or uridine. Included within the term aptamers aresynthetic aptamers that incorporate analogous forms of purines andpyrimidines. “Analogous” forms of purines and pyrimidines are thosegenerally known in the art, many of which are used as chemotherapeuticagents. Non-limiting examples of analogous forms of purines andpyrimidines (i.e. base analogues) include aziridinylcytosine,4-acetylcytosine, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N6-methyladenine, 7-methylguanine,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5-methoxyuracil,2-methyl-thio-N6-isopentenyladenine, uracil-5-oxyacetic acidmethylester, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid, 5-pentynyl-uracil, and 2,6-diaminopurine. Theuse of uracil as a substitute base for thymine in deoxyribonucleic acid(hereinafter referred to as “dU”) is considered to be an “analogous”form of pyrimidine in this invention.

Aptamer oligonucleotides may contain analogous forms of ribose ordeoxyribose sugars that are known in the art, including but not limitedto 2′ substituted sugars such as 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars,epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars,furanose sugars, sedoheptuloses, locked nucleic acids (LNA), peptidenucleic acid (PNA), acyclic analogs and abasic nucleoside analogs suchas methyl riboside.

Aptamers may also include intermediates in their synthesis. For example,any of the hydroxyl groups ordinarily present may be replaced byphosphonate groups, phosphate groups, protected by a standard protectinggroup, or activated to prepare additional linkages to additionalnucleotides or substrates. The 5′ terminal OH is conventionally free butmay be phosphorylated; OH substituents at the 3′ terminus may also bephosphorylated. The hydroxyls may also be derivatized to standardprotecting groups. One or more phosphodiester linkages may be replacedby alternative linking groups. These alternative linking groups include,but are not limited to embodiments wherein P(O)O is replaced by P(O)S(“thioate”), P(S)S (“dithioate”), P(O)NR 2 (“amidate”), P(O)R, P(O)OR′,CO or CH 2 (“formacetal”), wherein each R or R′ is independently H orsubstituted or unsubstituted alkyl (1-20 C.) optionally containing anether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl.

One particular embodiment of aptamers that are useful in the presentinvention is based on RNA aptamers as disclosed in U.S. Pat. Nos.5,270,163 and 5,475,096, which are incorporated herein by reference. Theaforementioned patents disclose the SELEX method, which involvesselection from a mixture of candidate oligonucleotides and stepwiseiterations of binding, partitioning and amplification, using the samegeneral selection scheme, to achieve virtually any desired criterion ofbinding affinity and selectivity. Starting from a mixture of nucleicacids, preferably comprising a segment of randomized sequence, the SELEXmethod includes steps of contacting the mixture with a target, such as atarget analyte, under conditions favorable for binding, partitioningunbound nucleic acids from those nucleic acids which have boundspecifically to target molecules, dissociating the nucleic acid-targetcomplexes, amplifying the nucleic acids dissociated from the nucleicacid-target complexes to yield a ligand-enriched mixture of nucleicacids, then reiterating the steps of binding, partitioning, dissociatingand amplifying through as many cycles as desired to yield highlyspecific, high affinity nucleic acid ligands to the target molecule. Insome embodiments, negative screening is employed in which a plurality ofaptamers are exposed to analytes or other materials likely to be foundtogether with target analytes in a sample to be analyzed, and onlyaptamers that do not bind are retained.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. In some embodiments, two or more aptamers are joined toform a single, multivalent aptamer molecule. Multivalent aptamermolecules can comprise multiple copies of an aptamer, each copytargeting the same analyte, two or more different aptamers targetingdifferent analytes, or combinations of these.

Aptamers can be used as diagnostic and prognostic reagents, as reagentsfor the discovery of novel therapeutics, as reagents for monitoring drugresponse in individuals, and as reagents for the discovery of noveltherapeutic targets. Aptamers can be used to detect, modify the functionof, or interfere with or inhibit the function of one or more targetanalytes. The term “analytes” as used herein includes without limitationdrugs, prodrugs, pharmaceutical agents, drug metabolites, biomarkerssuch as expressed proteins and cell markers, antibodies, serum proteins,cholesterol and other metabolites, electrolytes, metal ions,polysaccharides, nucleic acids, biological analytes, biomarkers, genes,proteins, hormones, or any combination thereof. Analytes can becombinations of polypeptides, glycoproteins, polysaccharides, lipids,and nucleic acids. Aptamers can inhibit the function of gene products byany one of, but not limited to only, the following mechanisms: (i)modulating the affinity of a protein-protein interaction; (ii)modulating the expression of a protein on a transcriptional level; (iii)modulating the expression of a protein on a post-transcriptional level;(iv) modulating the activity of a protein; and (v) modulating thelocation of a protein. The precise mechanism of action of peptideaptamers can be determined by biochemical and genetic means to ascertaintheir specific function in the context of their interaction with othergenes, and gene products.

Aptamers can be used to detect an analyte in any of the detectionschemes described herein. In one embodiment, apatamers are covalently ornon-covalently coupled to a substrate. Non-limiting examples ofsubstrates to which aptamers may be coupled include microarrays,microbeads, pipette tips, sample transfer devices, cuvettes, capillaryor other tubes, reaction chambers, or any other suitable formatcompatible with the subject detection system. Biochip microarrayproduction can employ various semiconductor fabrication techniques, suchas solid phase chemistry, combinatorial chemistry, molecular biology,and robotics. One process typically used is a photolithographicmanufacturing process for producing microarrays with millions of probeson a single chip. Alternatively, if the probes are pre-synthesized, theycan be attached to an array surface using techniques such asmicro-channel pumping, “ink-jet” spotting, template-stamping, orphotocrosslinking. An exemplary photolithographic process begins bycoating a quartz wafer with a light-sensitive chemical compound toprevent coupling between the quartz wafer and the first nucleotide ofthe DNA probe being created. A lithographic mask is used to eitherinhibit or permit the transmission of light onto specific locations ofthe wafer surface. The surface is then contacted with a solution whichmay contain adenine, thymine, cytosine, or guanine, and coupling occursonly in those regions on the glass that have been deprotected throughillumination. The coupled nucleotide bears a light-sensitive protectinggroup, allowing the cycle can be repeated. In this manner, themicroarray is created as the probes are synthesized via repeated cyclesof deprotection and coupling. The process may be repeated until theprobes reach their full length. Commercially available arrays aretypically manufactured at a density of over 1.3 million unique featuresper array. Depending on the demands of the experiment and the number ofprobes required per array, each wafer, can be cut into tens or hundredsof individual arrays.

Other methods may be used to produce the biochip. The biochip may be aLangmuir-Bodgett functionalized glass, germanium, silicon, PTFE,polystyrene, gallium arsenide, gold, silver, membrane, nylon, PVP, orany other material known in the art that is capable of having functionalgroups such as amino, carboxyl, Diels-Alder reactants, thiol or hydroxylincorporated on its surface. These groups may then be covalentlyattached to crosslinking agents, so that the subsequent attachment ofthe nucleic acid ligands and their interaction with target moleculeswill occur in solution without hindrance from the biochip. Typicalcrosslinking groups include ethylene glycol oligomer, diamines, andamino acids. Alternatively, aptamers may be coupled to an array usingenzymatic procedures, such as described in US20100240544.

In some embodiments, aptamers are coupled to the surface of a microbead.Microbeads useful in coupling to oligonucleotides are known in the art,and include magnetic, magnetizable, and non-magnetic beads. Microbeadscan be labeled with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dyes tofacilitate coding of the beads and identification of an aptamer joinedthereto. Coding of microbeads can be used to distinguish at least 10,50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 5000,or more different microbeads in a single assay, each microbeadcorresponding to a different aptamer with specificity for a differentanalyte.

In some embodiments, reagents are coupled to the surface of a reactionchamber, such as a tip. For example, the interior surface of a tip maybe coated with an aptamer specific for a single analyte. Alternatively,the interior surface of a tip may be coated with two or more differentaptamers specific for different analytes. When two or more differentaptamers are coupled to the same interior tip surface, each of thedifferent aptamers may be coupled at different known locations, such asforming distinct ordered rings or bands at different positions along theaxis of a tip. In this case, multiple different analytes may be analyzedin the same sample by drawing a sample up a tip and allowing analytescontained in the sample to bind with the aptamers coated at successivepositions along the tip. Binding events can then be visualized asdescribed herein, with the location of each band in a banding patterncorresponding to a specific known analyte.

In some embodiments, binding of one or more aptamers to one or moretarget analytes is detected using an optical feature. In someembodiments, the optical feature is fluorescence. In some embodiments, asample containing analytes to be analyzed is treated with a labelingcompound to conjugate the analytes with a fluorescent tag. Binding canthen be measured by fluorescence to detect presence and optionallyquantity of one or more analytes, such as illustrated in FIG. 136 incombination with aptamers coupled to an array, and in FIG. 137 incombination with aptamers coupled to coded beads. In some embodiments,the sample is treated with a labeling compound to conjugate the analyteswith a linker. Upon binding the linker is functionalized with afluorescent tag and the positive event is measured by fluorescence. Insome embodiments, the analyte binding domain of an aptamer is partiallyhybridized to a complentary probe that is fluorescently labeled. Uponbinding to the analyte, the complementary probe is released, whichresults in an optically measurable decrease in fluorescent signal. Insome embodiments, an aptamer is fluorescently labeled and is partiallyhybridized to a complementary probe labeled with a quencher that is inproximity to the fluorescent label. Upon binding to the analyte, thecomplementary probe is released resulting in a measurable increase influorescence of the label conjugated to the aptamer. In someembodiments, the aptamer is partially hybridized to a complementaryprobe, which hybridization occludes a domain containing a secondarystructure. Upon binding to the analyte, the complementary probe isreleased, and the secondary structure is made available to anintercalating dye used to produce a measurable signal. Labels useful inthe detection of binding between an apatamer and an analyte in a bindingpair can include, for example, fluorescein, tetramethylrhodamine, TexasRed, or any other fluorescent molecule known in the art. The level oflabel detected at each address on the biochip will then vary with theamount of target analyte in the mixture being assayed.

In some embodiments, the displaced complementary probe is conjugated toone member of an affinity pair, such as biotin. A detectable molecule isthen conjugated to the other member of the affinity pair, for exampleavidin. After the test mixture is applied to the biochip, the conjugateddetectable molecule is added. The amount of detectable molecule at eachsite on the biochip will vary inversely with the amount of targetmolecule present in the test mixture. In another embodiment, thedisplaced complementary probe will be biotin labeled, and can bedetected by addition of fluorescently labeled avidin; the avidin itselfwill then be linked to another fluorescently labeled, biotin-conjugatedcompound. The biotin group on the displaced oligonucleotide can also beused to bind an avidin-linked reporter enzyme; the enzyme will thencatalyze a reaction leading to the deposition of a detectable compound.Alternatively, the reporter enzyme will catalyze the production of aninsoluble product that will locally quench the fluorescence of anintrinsically-fluorescent biochip. In another embodiment of thedisplacement assay, the displaced complementary probe will be labeledwith an immunologically-detectable probe, such as digoxigenin. Thedisplaced complementary probe will then be bound by a first set ofantibodies that specifically recognize the probe. These first antibodieswill then be recognized and bound by a second set of antibodies that arefluorescently labeled or conjugated to a reporter enzyme. Manyvariations on these examples are known or will now occur to thoseskilled in the art. Assays analogous to “double-sandwich” ELISAs canalso be set up using combinations of antibodies and aptamers asreceptors. For example, a capture surface can be functionalized with anaptamer and the detection reagent can be an enzyme-labeled antibody.Conversely, the antibody can be on the capture surface and the detectionreagent a labeled aptamer.

In some embodiments, a sample containing an anlalyte to be analyzed isdispersed into a three-dimensional hydrogel matrix. The hydrogel matrixcan be activated to covalenly trap proteins and small molecules. After awash of the excess and unbound sample, fluorescently labeled aptamerscan be introduced for the detection of the specific analytes present,such as illustrated in FIG. 138. In some embodiments, thethree-dimensional hydrogel matrix is divided in small subsets ormicrowells to which a single aptamer can be added to undergo a specificanalysis of the analyte present. In some embodiments, aptamers arelabeled with a set of coded quantum dots or fluorescent tagscorresponding to a unique signature. In some embodiments, labeledaptamers are added to the three-dimensional matrix simultaneously withthe sample.

In some embodiments, an aptamer is used instead of an antibody in anELISA assay. In general, a sample is exposed to a surface andspecifically or non-specifically coupled thereto. In a sandwich ELISA,an analyte is specifically coupled to a surface by binding to firstantibody that is coupled to the surface. In a typical ELISA, theanalyte, whether bound specifically or non-specifically, is thendetected by binding to a second antibody carrying a label. In an aptamerELISA, the first antibody, second antibody, or both are replaced withaptamers specific for an analyte.

Imaging Analysis of Samples and Assay Reaction Products

In some embodiments of the invention, analysis of sample and the assayreaction products can be performed using digital imaging. The assaycuvettes can be aligned for measurement and scanned or imaged in asingle operation. In the instrumented system of the invention this isachieved automatically by mechanical components. Assay cuvettes arelocated at defined locations in a cartridge and moved to the scannermaintaining the same orientation and spacing. The graph shown in FIG. 92corresponds to the green channel response over the width of the cuvette.As shown, the edges of the cuvettes are well-defined, as is the locationcorresponding to the middle of the cuvette.

The images obtained by scanning or imaging can be a two-dimensionalarray of pixels, where each pixel comprises a plurality of intensityvalues corresponding to a distinct detection spectral region (e.g., red,blue, green). The images can be interpreted by line-scans, which maycorrespond to a horizontal portion of a tip. If the tip iscircular-shaped, then an effective absorbance can be determined bydeconvoluting the line-scan over an appropriate function. Examplefunctions include parabolic functions, and functions for circles. Insome embodiments, the images can be data-averaged over multiple imagestaken of a tip or a sample over a range of physical locations.

In an embodiment, a sensor is provided to locate an assay unit relativeto a detector when an assay is detected.

As shown in FIG. 61 and FIG. 62, bromophenol blue solutions wereaspirated into a set of conical tips and imaged with front faceillumination (light source and detector on the same side of the object).Small volumes (5 uL) of serial dilutions of a 0.78 mg/mL solution wereused with the highest concentration at the top of the image. In FIG. 61,tips on the left have the sample located at the widest location in theconical tip whereas tips on the right have the sample at the narrowest.The image in FIG. 61 was taken using a scanning optical system.

FIG. 62 shows tips that were imaged using a back-lit configuration(light source and detector on opposite sides of the imaged object). Theback-lit configuration can be preferred because of the higher imagequality.

As shown in FIG. 61 and FIG. 62, the effective optical path length of acolored solution can be varied by changing tip design. In particular,the pathlength can be varied within a single tip to increase sensitivityof measurement of light absorbance (long pathlength) or to increase thedynamic range of the measurement. The pathlength can be changed, forexample, by changing the diameter of the tip.

An additional feature of the tip design can be that it enables assays tobe read with a very small volume of assay reaction product requiring avery small volume of sample. Typically, assay reaction mixtures areincubated in a narrow part of a tip which provides a high ratio ofliquid/air surface area to volume, thus minimizing evaporation. Thesmall volume can then be moved to a wide part of the tip for measurementof the colored product thus maximizing the optical pathlength available(and thereby increasing the absorbance of light) for a given reactionmixture volume.

For example in the table below, we compare reading an assay reactionmixture of 10 uL in which a 1 uL sample is diluted 1:10. In the tips ofthe current invention, incubation of an assay mixture can be achieved ina 13 mm length of tip region having a diameter of 1 mm then be moved toa 3 mm diameter region for color measurement. In comparison with using amicrotiter plate of standard dimensions (typical of 384-well plates) toincubate and read the same assay, the area of liquid surface exposed toair (allowing evaporation) is about 5 times less and the opticalpathlength is about twice as great.

Sample volume 1.00 uL Dilution factor 10.00 fold Reaction volume 10.00uL Tips Tip diameter 1.00 mm For incubation Exposed surface area 1.57mm{circumflex over ( )}2 For incubation Length of liquid column 12.73 mmFor incubation Tip diameter 3.00 Pathlength for reading Length of liquidcolumn 1.41 mm For reading Microtiter Plate Well diameter 3.00 mmExposed surface area 7.07 mm{circumflex over ( )}2 Length of liquidcolumn 1.41 mm Pathlength

Optimizing Optical Path Length

Spectroscopic measurements of colored solutes are traditionally measuredby recording the fraction of light transmitted through a cuvette at theabsorbance wavelength maximum. The data are then transformed to giveAbsorbance (A) or optical density (OD) values. According to Beer's law,A(λmax)=εM*1*Concentration where εM is the molar extinction product(L/Mole.cm), 1 is the optical pathlength (cm) and Concentration is inmolar units. OD=A for 1=1. This is done to provide a measure, A, whichis directly proportional to solute concentration.

There are two significant limitations of absorbance measurements forassaying solute concentrations. At low concentrations, the change intransmission is small and therefore imprecise because of variations inthe background (or blank) transmission. At high concentrationstransmission is very low (for example at A=3, the transmitted light is1/1000 th of the input light. Any “stray” light or other forms of signalnoise have a significant effect on the measurement and the response toconcentration becomes non-linear and imprecise. Typically, absorbancemeasurements are regarded as precise and accurate over a range fromabout 0.1 to about 2.0 (a 20-fold range).

The method of the present invention overcomes these problems to asignificant degree by enabling facile measurements of color over a verywide dynamic range (up to 1000-fold):

1. At different pathlengths: low concentrations can be measured at longpathlengths and high concentrations at short pathlengths.

2. In different color channels: low concentrations can be measured inthe best matching color channel and high concentrations in colorchannels mismatched to the color.

This is illustrated by the data shown in FIG. 79. Bromphenol bluesolutions serially diluted from a 5 mg/mL stock were analyzed using thethree-color method in tips at two locations, one with a maximumpathlength (also “path length” herein) of about 5 mm (“wide”), the otherof about 1 mm (“narrow”). Signals in the three color channels werenormalized to their highest and lowest levels as shown in the graphbelow. An algorithm to optimally extract the concentration of theanalyte (bromphenol blue) was set up as follows:

1. For normalized signals in the range 10% maximum<signal<90% maximum,compute a value concentration=a+b*Log(signal)+c*(Log(signal)){circumflexover ( )}2 where a b and c are arbitrary constants. This operation wasperformed for each color at both pathlengths.

2. Using a well-known optimization routine (for example “Solver” inMicrosoft Excel), compute the best-fit values of a, b and c for allcolors and pathlengths.

3. Average the computed concentration values for all colors and bothpathlengths.

As shown in FIG. 80, the method yielded accurate results across a1000-fold concentration range. When the algorithm was used to computeconcentration values for replicate measurements (N=3), the average CVwas 3.5%.

Measurements can be made at various pathlengths. In some cases,pathlengths are at least partially dependent on container (e.g.,cuvette, tip, vial) geometry. The container geometry and/or features inthe container, such as scattering features, may affect the optical pathand path length in the container.

Multi-Color Analysis

Scanners and cameras have detectors that can measure a plurality ofdifferent colors channel detection spectrum regions (e.g., red, green,and blue). Because the spectral width of each of these channels is wideand color chemistries produce colored products with wide band widths,colored reaction products can be detected using a plurality of channeldetection spectrums. For example, FIG. 71 shows the response of red(squares), green (diamonds), and blue (triangles) detection channelspectrums as a function of analyte concentration. The signals producedby each detector correspond to light intensity within each detectionspectrum and are typically expressed as a number from 0 to 255. Whenwhite light is transmitted through a circular section cuvette containinga colored solute as shown above, light is absorbed and the lightintensity reduced so that the detector responses change.

For example, when bromophenol blue dissolved in alkaline buffer atconcentrations ranging from 0 to 5 mg/mL and scanned at the locationindicated “C3” in FIG. 62, signals shown in FIG. 66, which are thedetector responses averaged over a zone corresponding to seven pixelsalong the length of the cuvette. The signals were recorded on an Epsonbacklit scanner. FIG. 66 shows the three color responses for a set of 11cuvettes containing 2-fold serial dilutions of a 5 mg/mL bromophenolblue solution and a “blank” solution (arranged left to right on theimage). The image of the scanned tips is shown in FIG. 67. The signal ineach channel corresponding to the solution is reduced to an extentrelated to the optical path. Accordingly, the maximum change in signalis seen at the center of the cuvette. When signals in the central regionof the cuvette were averaged (over the zone shown by the smallrectangles for the fourth cuvette from the left) and plotted against thebromophenol blue concentration, the dose-responses shown FIG. 68 wereobserved. In each color “channel” the signal declined smoothly withconcentration. The green signal changed most and the blue signal least.Corresponding optical densities measured in an M5 spectrometer(Molecular Devices) at the wavelength of maximal absorbance (e.g., 589nm) are also shown. At the highest concentrations, the spectrophotometerresponse becomes not linear and changes very little with concentration.A similar effect was noted in the scanner green and red channelresponses. The blue channel response in contrast, is very slight untilthe highest concentrations.

According to Beer's law, absorbance of a solution is equal toεM*Concentration*pathlength. Absorbance is defined as Log10(Transmission/Blank Transmission), where blank transmission is thatcorresponding to that for the solvent. Strictly Beer's law applies to aparallel beam of monochromatic light (in practice a band width of a fewnm) passing normally through a rectangular cuvette. Spectrophotometersrespond linearly to concentration up to Absorbance values about 1.5. Athigher absorbance, instrument response becomes non-linear due to “straylight” and other effects. Optical density is defined as absorbance for aone cm optical pathlength.

When the color signal data from the above experiment was transformedaccording to an expression that linearizes optical transmission so as toobtain an absorbance value proportional to concentration in conventionalspectrophotometry (−Log(signal/blank signal), the graph shown in FIG. 69was obtained for the green (squares) and red (diamonds) channels.

The green channel data followed Beer's law but the red channel data didnot reaching a plateau level at for a sample having about 2 mg/mL in afashion similar to that of the OD response of the spectrophotometer.

Improved Assay Utilization by Three-Color Analysis and Optimization ofOptical Path Length

Assay results from reaction setups that would otherwise provideuninterpretable data can be salvaged using the present invention. Thepresent invention allows for increased dynamic range and sensitivity ofassays by the combination of optical pathlength optimization andthree-color analysis. The inability to salvage data plagued by reduceddynamic range is a major problem in assay management, especially in thecontext of samples being evaluated for diagnostic or therapy managementpurposes is that assays have a limited dynamic range or limited range ofanalyte values that can be reported with good confidence. There are twomain reasons why an assay result may not be available fromlaboratory-based assay systems or from distributed test situations.Namely the analyte value is too high or too low to be reported. This mayin some circumstances be rectified in clinical laboratories byre-analyzing a portion of a retained sample using a different dilution.In distributed testing typically there is no recourse but to recall thepatient, obtain a new sample and use a different (laboratory) method.This is because assay systems use fixed protocols and fixed levels ofsample dilution. In either situation, it is very inconvenient andexpensive to rectify the problem. Moreover, valuable informationpertinent to proper diagnosis and/or therapy management may be lost withresultant harm to the patient.

In the system of the present invention, these problems are eliminated bymonitoring assays during their execution, recognizing any problem andmodifying either the optical pathlength used to measure the assayproduct or making use of the different sensitivity levels of the threecolor channels to the assay color and in turn to the analytesensitivity.

Specifically when the assay reaction product is measured if the measuredsignal is either too high or too low, the system can respond by:

1. making the measurement with a different pathlength (moving theoptical cuvette relative to the optical system such that the pathlengthis either bigger or smaller). This can be performed by (a) making ameasurement at a standard, first location, (b) reporting the result tothe software managing the assay (in instrument and/or on a remoteserver), (c) recognizing a problem condition, and (d) modifying the readposition and making a second measurement; and/or

2. emphasizing a more or less sensitive color channel in signalanalysis. This can be implemented automatically by suitable assayanalysis algorithms.

Color Calibration

The signal responses can be calibrated to allow for computation of theconcentration of the colored species from imaging data. To obtain a datatransform predictive of the concentration of the colored solute, thefollowing procedure can be used. In other embodiments, other methods mayalso be used.

1. For each channel for all concentrations, the transform−Log(signal/blank signal) was computed and designated “A”.

2. For all concentrations, a further transform (“C”) was computed asa*A+b*A{circumflex over ( )}2+c*A{circumflex over ( )}3 (initiallyvalues for a, b and c were set at arbitrary values).

3. For all concentrations, C values for the three color channels weresummed and designated Cestimate.

4. The sum of square differences between the target (known)concentration and Cestimate was computed over all concentrations.

5. Values of a, b and c parameters for all channels were derived by awell-known algorithm which minimized the sum of the square differences.

The results shown in FIG. 70 demonstrates accurate calibration of thescanner response over the entire concentration range.

Other automated calibration algorithms have been developed and found tobe equally effective. For example, the following is an example ofcalibration for a cholesterol assay performed in a reaction tip.

The measured signal is decomposed into Red (R), Green (G), and Blue (B)color channels. Calibration equations are computed to optimize theaccuracy, precision, and dynamic range according to assay designrequirements.

In this assay example, only Red and Green channels are utilized tocompute concentration. These two signals are transformed to compute anintermediate variable (F) as follows:

F=p ₁ +p ₂ ·G+p ₃ ·G ² +p ₄ ·R+p ₅ ·R ²

where p₁ are calibration parameters.

Finally, the signal F is used to compute the concentration (C) via alinear transformation:

C=(F−p ₆)/p ₇,

where C is the calculated concentration, and p₆ and p₇ are calibrationparameters, in this case, representing the intercept and slopeparameters of a linear relationship, respectively.

When the same approach was followed for a large set of assays for avariety of analytes which produced colored products spanning the entirevisible spectrum (max from 400-700 nm), comparable results wereobtained.

In conventional transmission spectrophotometric measurements, a “blank”value is used to normalize the measurement. Method (1) Blanks aretypically constructed by measuring a sample that is equivalent to thesample but does not have any of the component to be measured. Themeasurement is typically made in the same cuvette as that which will beused for the sample or an optically equivalent cuvette. Thus in aspectrophotometric assay, one would combine all the reagents in the sameconcentrations using the same protocol substituting a zero analytesolution for the sample. Method (2) uses a two step process makingmeasurements against an absolute reference such as air (which will nevervary in absorbance) and measuring both sample and blank against theabsolute reference. The sample absorbance is then calculated bysubtraction of the blank value from that of the sample. Method (3) is tocollect spectra of the sample or assay reaction product and referencethe measured absorbance (or transmission) at an optimal wavelength(usually that for maximum absorbance for the measured species) againstthe absorbance at a wavelength where the species to be measured is knownto have zero absorbance. The absorbance is the difference between thoserecorded at the two wavelengths.

Digital imaging and three-color analysis can be employed, but in someembodiments can be modified according to the digital (pixilated)character of the assay signal. Namely:

1. For each pixel in the image and for each color a white standard isimaged and the intensities of the signal adjusted to a valuecorresponding to no absorbance. This can be done by the followingexemplary procedure:

-   -   a. adjusting the intensity of the light source    -   b. adjusting the sensitivity of the detector (preferred), or    -   c. software adjustment (not preferred by itself)

A preferred approach is a combination of (b) and (c) above. First,adjust the detector in the analog realm, and then fine tune the resultin the digital realm.

For the analog adjustment, the gain and offset of the amplifiers betweenthe light sensors and the analog-to-digital section are adjusted toensure maximum resolution of the digitization. The lower end of thelight range of interest will be set to zero and the high end of therange will be set to just below saturation of the sensor.

Subsequently, the images may be fine-tuned in the digital domain. Apreferred approach, specifically, would be to use what is called the“two-image calibration” for an m×n image. The mechanism is to firstcollect a black image by blocking all light to the detector. We'll callthis image BLACK[m,n]. A second calibration image is recorded consistingof light at the maximum end of the sensitivity range. We'll call thisimage WHITE[m,n]. Thus a corrected image a[m,n] could be constructed,pixel-wise, as:

${a\left\lbrack {m,n} \right\rbrack} = \frac{{c\left\lbrack {m,n} \right\rbrack} - {{BLACK}\left\lbrack {m,n} \right\rbrack}}{{{WHITE}\left\lbrack {m,n} \right\rbrack} - {{BLACK}\left\lbrack {m,n} \right\rbrack}}$

Note that this digital correction does not improve the dynamic range ofthe digitized data, but adjusts the values so that the full white andblack references are consistent.

2. An image of a physical blank in a tip can be used as a pixel-by-pixeland color by color blank. The blank can be:

-   -   a. Air;    -   b. Water;    -   c. Blank assay reaction product (no analyte);    -   d. Sample blank (no assay reagents); or    -   e. Some combination of the above;

3. The signal from a color channel where there is a zero or weakresponse can be used to normalize signals from the other channels.

A further method of controlling and normalizing the optics is to image aset of physical (stable) standards before or during an assay. Forexample, an array of printed dyes (shown in FIG. 104) can be madecorresponding to a set of standard colors with standard intensities(similar to standard color “wheels” used to calibrate cameras andscanners).

Such standards may be measured using reflectance from an opaque surfaceor (preferred) by transmission through a clear film.

Depending on the stability of the optics, calibration and normalizationof the optics may be (1) a one-time exercise, (2) performed at regularintervals or (3) performed for each assay.

Calibrating a Digital Imager Range

In some embodiments, methods may be provided for calibrating a digitalimager used for imaging optical densities.

In testing the optical density of an analyte, it may be desirable tomake use of as much of the dynamic range of the imager as possible.Under normal use, the setup may comprise a relatively homogenousilluminated white background, the imager and the analyte to be tested ina transparent cuvette between them. Operationally, the test may compriseplacing the cuvette between the imager and the white backlight sourceand measure the amount of light absorbed by the analyte in the cuvette.To maximize the full dynamic range of the sensor, the background may besensed as the maximum intensity measurable. It may be desirable to takecare to not saturate the sensor because then information could be lostsince when the sensor is saturated, and attenuation may not be correctlymeasured. The system may be configured to efficiently maximize themeasured values of the backlight while minimizing number of saturatedpixels.

The illuminated background may emit white light of equal intensity overits entire surface. The light output may vary somewhat, producing anormal distribution of pixel intensities as detected by the imager. Thisis illustrated by the curves shown in FIG. 128. For this example, thesensor may return a value from 0 to 256 from each pixel as an indicatorof the amount of light it receives. Each pixel may saturate at a valueof 256. That is, regardless of further increasing of light intensity orsensor sensitivity, only a value of 256 may be recorded. Series 1 inFIG. 128, the dotted line, shows where the light is too intense, cuttingoff the normal curve. Series 3, the dashed line, shows that all pixelsare correctly reading intensity, but that the imager sensitivity islower than it might be for maximum dynamic range. The majority of thepixels are at a value of less than 200. Series 2 represents the desiredsettings, where the mean of the distribution is as high as possible, butthat a sufficiently small number of pixels are saturated.

In one embodiment, the intensity of the backlight may be held constantwhile the imager's settings may be adjusted. For the purpose of imagersensitivity, two controls may be used: exposure time and gain. Exposuretime may be the amount of time that the sensor pixels are permitted tocollect photons before the value is read out. For a given amount oflight, the readout value may be larger when the exposure time is madelonger. This control may be the “coarse” control for the application.Gain may be the control adjusting the amount of amplification applied tothe sensor signal. Increasing gain may increase the value of the signalfrom the sensor. Gain may be the “fine” control.

An exemplary procedure for setting the imager's sensitivity parametersmay include one or more of the following steps:

-   -   1. Set exposure time to value known to be below saturation. Set        gain to highest usable value.    -   2. Binary search starting upwards adjust exposure time to find        the setting where not all of the pixels in the region of        interest of the image are saturated. This may be detected by        observing the point at which the mean pixel value becomes less        than 256.    -   3. Back gain down incrementally until there are sufficiently few        pixels that are at the saturation limit. The number of pixels at        an acceptable level will be determined by the shape of the        distribution. Wide standard deviation will increase the number        of pixels permitted to be saturated.

Next, the white balance may be corrected. There are three groups ofsensors in a digital imager. Members of each group collect light of adifferent wavelength, red, green or blue. When detecting white light,the sensors would preferably see equal values or red, green and blue.The white balance control adjusts the relative gains of the red and bluechannel. Since the light coming from the backlight is defined as white,the procedure would be to simply adjust the white balance until thechannels read the same values. In practice, the green channel istypically left unadjusted, and the red and blue channels are changed inopposite directions to each other as the control is changed. However, inother embodiments, another channel, such as the red channel or bluechannel may be left unadjusted while the other two channels may bechanged.

Finally, the images may be fine-tuned in the digital domain A preferableapproach, specifically, would be to use what is called the “two-imagecalibration” for an m×n image, as previously described.

Assays making a variety of colored products have been analyzed in thesubject invention. Colors from those with low wavelength absorptionmaxima (yellow) to high wavelength maxima (blue) have been successfullymeasured. Wavelength maxima for some representative assays were: 405,450, 500, 510, 540, 570, 612 and 620 nm demonstrating the ability toread color over the entire visible spectrum.

Colors may be quantified using average data for many pixels (typicallyabout 1000). A parameter (f) which produces a good fit (e.g., greatestR²) to the dose-response data may be selected. The parameter may befirst fitted to the form a1+b1*R+c1*R²+b2*G+c2*G²+b3*B+c2*B² where a, b,c are constants and R, G and B are color intensity values for red, greenand blue channels respectively. The parameter f may then be derived byforcing it to have a maximum value of 1 and a minimum value of 0.Parameter f is related to transmission of light through the coloredreaction product. As would be expected, f may be closely related to theparameter optical density (OD) used in spectrophotometry to quantify anabsorbing species. When 1—f measured by 3-color imaging is plottedagainst OD measured at the absorption maximum for the same assayreaction products in a microtiterplate in a spectrophotometer, it may beobserved that 1—f is essentially linearly related to OD. In FIG. 129,such data for five assays is presented. OD may be normalized as“relative OD”=(OD−OD min)/(ODmax−OD min). In some cases, there is asomewhat curved relationship but the correlation coefficient (R) isusually >0.99.

The parameter f may be used to calibrate assays measured by 3-colorimage analysis. When plotted against concentration of the analyte, asmooth calibration relationship may be shown in FIG. 130 for arepresentative cholesterol assay. An equation of the formconcentration=a+b*f+c*f² (where a, b and c are constants) relatingconcentration to f is derived and as shown in FIG. 130, the calculatedconcentration is essentially identical to that of the “nominal”(expected, desired) value (regression line slope close to 1.0, interceptclose to 0.0 and R²=0.998. Also shown in FIG. 130 are graphs of assayaccuracy and precision. Accuracy is close to 100% (mean 100.2%) andimprecision (represented by CV %) is low (less than 10%, average CV3.9%).

Simultaneous Imaging of Assays

As shown in FIG. 56, FIG. 57, FIG. 58, FIG. 59, and FIG. 60, severalassay elements (tips, wells, blots) can be imaged in parallel. Ingeneral, the elements can be placed at known locations in a cartridge ormounted on a subsystem of the instrument, so that a particular elementcan be associated with a particular assay. Even if the elements are notperfectly oriented or located, image analysis can be used to rectify anysuch miss-positioning by locating features of the assay elements.

Commercially available assays for albumin (FIG. 56) and cholesterol(FIG. 57) were used according to the manufacturer's directions. A seriesof analyte concentrations in the range of clinical interest was measuredusing a series of calibrators in which the analyte concentration wasreduced two-fold from the highest concentration. In FIG. 56 and FIG. 57,analyte concentration was highest on the right and the furthest left tipcorresponded to zero analyte. The volume of assay reaction mixtureaspirated into the tips was 20 uL.

FIG. 58, FIG. 59, and FIG. 60 show wells that can be imaged in parallel.A set of shallow hemispherical wells was made by machining a block ofwhite opaque plastic. Three commercially available color forming assayswere performed in these wells and reaction products imaged. As above,the wells to the far right have the highest analyte concentration andeach adjacent well has a two-fold lower concentration except theleft-most well which has zero analyte. Seven uL of assay reactionproduct were introduced into each well.

Reaction products can also be imaged after blotting them onto porousmembranes or paper and imaging once the liquid has soaked into themedium. It is also possible to use any of a variety of assay chemistriesimpregnated into paper or membranes and to image the resulting reactionproducts following addition of sample.

Analyzing Turbidity

Turbidimetry is performed by measuring the reduction in the intensity ofthe incident light after it passes through the sample being measured.This technique is used where the result of the assay is a dispersedprecipitate that increases the opacity of the liquid.

Turbidimetry can be measured in latex agglutination assays. As a modelof latex agglutination assay responses, polystyrene latex particles (1um diameter) were dispersed in buffer at the given (w/v) concentrationsand subject to three-color image analysis. As can be seen in FIG. 72, agood response was found in all three channels and could be used tomeasure the latex particle concentration and agglutination of latex.

Analyzing Agglutination

Similarly to turbidity analysis, the system can be used to measureagglutination, hemagglutination, and the inhibition thereof.

The system can be used to perform blood typing by red blood cellagglutination. Blood was diluted and mixed with blood typing reagents(anti-A, anti-B, anti-D) from a commercial typing kit. As shown belowfor a B+ blood, the appropriate agglutination responses can easily beseen when the mixtures are imaged. Moreover, when the images shown inFIG. 77 were scanned along the vertical axis of the tips, a quantitativemeasure of agglutination could be obtained by measuring the variance ofthe three-color signals, as shown in FIG. 78. Greater variance indicatedagglutination and can be detected in each color channel. It is evidentthat the method can be used to measure the extent of such agglutinationreactions.

Shape Recognition

Images can be analyzed for shape recognition. Shape recognition can beperformed at normal magnification and at very high magnification. Underhigh magnification image analysis may be used to recognize the size andshape of cells. These techniques are commonly used in cell counting todetermine relative concentrations of red blood cells, white blood cellsand platelets. Under normal magnification, shape recognition is used toobserve the state of the sample. Bubble and other defect recognitionmethods are used to ensure that measured liquid amounts are aspiratedand dispensed correctly.

Analyzing Samples on Solid Phase Substrates

Digital imaging with front-face illumination can also be used to readout assay responses on solid phase substrates as shown in FIG. 76.Solutions of potassium chloride (0, 2, 4 and 8 mM) were added toReflotron™ potassium assay strips (Boehringer-Mannheim/Roche) designedfor use in a reflectance assay system.

Analyzing Sample Quality

Certain sample characteristics can render assay results invalid. Forexample, hemolysis causes potassium ions to leak from red cells intoplasma causing the measured plasma or serum potassium ion concentrationsto be falsely high. Similarly, icteria and lipemia can interfere withseveral color-forming chemistries by altering the measured absorbances.In the present invention, we can detect and quantify such interferingsubstances using image analysis. Assays which would give false resultscan then be either (1) eliminated from the list of results delivered bythe analytical system or (2) optical signals can be corrected to accountfor the measured level of interferent. An image of different types ofserum samples is shown in FIG. 99 (from left to right: Hemolyzed,Lipemic, Icteric (yellow) and “normal”).

Digital Data Analysis

Conventional methods for data generation and calibration in assaymethods which generate and/or change color typically measure an analogsignal representing the change in absorbance characteristics of an assaymixture generated by mixing a sample with reagents. Some portion of thereaction mixture is illuminated and the light transmitted through orreflected from that portion impinges on a detector and evaluated as ananalog signal. The quality of the assay as determined by the volume andquality of the sample, sample processing, assembly of the assay into theassay mixture and of the physical element used to present the mixture tothe optical system rely on an assumed quality of the physical systemused.

In the present invention, we can image (1) the sample, (2) sampleprocessing processes, and (3) the assay mixture and collect the data asa set of one or more digital images. Each pixel in the image of theassay mixture represents a very small fraction of the total but byaveraging the 3-color signal from many pixels, we collect an assaysignal at least as good as that obtained by conventional analog methods.Where however, conventional methods lose information by averaging, thepresent invention both aggregates the information and retains the detaillost by conventional methods. In this context, color-based assaysinclude assays for: Metabolites, Electrolytes, Enzymes, Biomarkers(using immunoassay), Drugs (using immunoassay), and Nucleic acid targets(using “LAMP” technology). The same principles can be applied to assaysusing fluorescence and/or luminescence.

Volume Confirmation and Correction

The volume of a sample, or any other material, such as a liquid or asolid, can be determined optically. This can be performed by imaging acontainer whose internal dimensions are known and mathematicallydetermining sample volume from observed segment of the containeroccupied. Solid measurements are primarily used to measure solids thatare centrifuged down. The most common case is reading the volume ofcentrifuged red blood cells to determining hematocrit level. Examples6-11 and 16 describe the use of imaging analysis to calculate samplevolumes and other measurements. This can allow for improved assayresults. For example, if the target volume to be used is 10 uL and thetechnology of the invention determines that the actual volume is 8 uL,the assay system can correct the results for the volume (in thisexample, the concentration of analytes calculated on the presumption ofa 10 uL sample would be multiplied by 10/8).

Knowledge of actual sample and reagent volumes can be performed byimaging the sample and reagents and can be used to correct thecalculations used to detect and/or quantify analytes in the sample.

As shown in many examples above, the use of imaging allows samples andassay mixtures to be evaluated for quality and assay response.Additionally, imaging of ‘tips” used as reaction vessels and sampleacquisition methods enables (1) the accurate and precise measurement ofsample and reagent volumes and (2) the use of such data to correct anyinaccuracies and or imprecision in assay results due to volume errors.To achieve this, tips can have accurately and precisely known geometry(as is the case for tips made by injection molding). Replicatemeasurements of tips using imaging has demonstrated that theirdimensions are precise to better than about 1%. It is thus possible tomeasure the volume of liquid samples and reagents in such tips withcorresponding precision. If the pipetting of samples and reagents isless accurate and precise, correction of results knowing the actualvolumes (by image measurement) is possible.

For example, consider an assay in which the response is directlyproportional to analyte concentration (as is true for many of the assaysdiscussed herein). A sample volume error of 10% would lead to an errorof 10% in the value reported by the analytical system. If however, theinaccurately dispensed sample volume is measured accurately (say towithin 2% of the actual value), the system response can be corrected soas to reduce the error from 10% to 2%. Corresponding corrections can bemade for volume errors in reagent volumes. The correction algorithm candepend on the response of the assay system to volume or knowledge ofeach assay component (sample, reagents), but this information can easilybe determined during assay development and validation.

Thus, the invention provides a variety of advantages over conventionaltechniques. In the generation of the “assay signal”, the presentinvention can detect physical defects in the assay cuvette, defects inthe assay mixture (bubbles and the like). Once these defects areidentified (image analysis) the assay result can be rejected so thatfalse results do not occur or (preferred) the effect of the defect canbe eliminated and an accurate assay signal computed.

In the assembly of the assay mixture, any and all defects can bedetected including: incorrect sample type (e.g. blood versus plasma),incorrect sample volume, for a blood sample, failure to separate plasmafrom formed elements (red and white cells), sample factors that maycompromise the quality of the assay result (e.g., lipemia, icteria,hemolysis, presence of precipitates, or other unidentifiedin-homogeneities), defects in assembly of the assay mixture (e.g.,presence of bubbles, failure to mix adequately (non-uniformity ofcolor)), mechanisms for retrospective quality evaluation andpreservation of detailed archival information, mechanisms for measuringsample and reagent volumes (and to correct for inaccuracies and/orimprecision in such volumes).

Assessing Therapeutic Agents

In a separate embodiment, devices and methods for monitoring more thanone pharmacological parameter useful for assessing efficacy and/ortoxicity of a therapeutic agent is provided. For example, a therapeuticagent can include any substances that have therapeutic utility and/orpotential. Such substances include but are not limited to biological orchemical compounds such as simple or complex organic or inorganicmolecules, peptides, proteins (e.g. antibodies) or a polynucleotides(e.g. anti-sense). A vast array of compounds can be synthesized, forexample polymers, such as polypeptides and polynucleotides, andsynthetic organic compounds based on various core structures, and thesecan also be included as therapeutic agents. In addition, various naturalsources can provide compounds for screening, such as plant or animalextracts, and the like. It should be understood, although not alwaysexplicitly stated that the agent is used alone or in combination withanother agent, having the same or different biological activity as theagents identified by the inventive screen. The agents and methods alsoare intended to be combined with other therapies. For example, smallmolecule drugs are often measured by mass-spectrometry which can beimprecise. ELISA (antibody-based) assays can be much more accurate andprecise.

Physiological parameters according to the present invention includewithout limitation parameters such as temperature, heart rate/pulse,blood pressure, and respiratory rate. Pharmacodynamic parameters includeconcentrations of biomarkers such as proteins, nucleic acids, cells, andcell markers. Biomarkers could be indicative of disease or could be aresult of the action of a drug. Pharmacokinetic (PK) parametersaccording to the present invention include without limitation drug anddrug metabolite concentration. Identifying and quantifying the PKparameters in real time from a sample volume is extremely desirable forproper safety and efficacy of drugs. If the drug and metaboliteconcentrations are outside a desired range and/or unexpected metabolitesare generated due to an unexpected reaction to the drug, immediateaction may be necessary to ensure the safety of the patient. Similarly,if any of the pharmacodynamic (PD) parameters fall outside the desiredrange during a treatment regime, immediate action may have to be takenas well.

Being able to monitor the rate of change of an analyte concentration orPD or PK parameters over a period of time in a single subject, orperforming trend analysis on the concentration, PD, or PK parameters,whether they are concentrations of drugs or their metabolites, can helpprevent potentially dangerous situations. For example, if glucose werethe analyte of interest, the concentration of glucose in a sample at agiven time as well as the rate of change of the glucose concentrationover a given period of time could be highly useful in predicting andavoiding, for example, hypoglycemic events. Such trend analysis haswidespread beneficial implications in drug dosing regimen. When multipledrugs and their metabolites are concerned, the ability to spot a trendand take proactive measures is often desirable.

In some embodiments, the present invention provides a business method ofassisting a clinician in providing an individualized medical treatment.A business method can comprise post prescription monitoring of drugtherapy by monitoring trends in biomarkers over time. The businessmethod can comprise collecting at least one pharmacological parameterfrom an individual receiving a medication, said collecting step iseffected by subjecting a sample of bodily fluid to reactants containedin a fluidic device, which is provided to said individual to yield adetectable signal indicative of said at least one pharmacologicalparameter; and cross referencing with the aid of a computer medicalrecords of said individual with the at least one pharmacologicalparameter of said individual, thereby assisting said clinician inproviding individualized medical treatment.

The devices, systems, and methods herein allow for automaticquantification of a pharmacological parameter of a patient as well asautomatic comparison of the parameter with, for example, the patient'smedical records which may include a history of the monitored parameter,or medical records of another group of subjects. Coupling real-timeanalyte monitoring with an external device which can store data as wellas perform any type of data processing or algorithm, for example,provides a device that can assist with typical patient care which caninclude, for example, comparing current patient data with past patientdata. Therefore, also provided herein is a business method whicheffectively performs at least part of the monitoring of a patient thatis currently performed by medical personnel.

Optical Setup for Sample and Reaction Product Imaging

Sample and reaction product analysis can be performed using an opticalsetup. The optical setup can includes a light source, an aperture, and asensor or a detector. A schematic for an optical setup is shown in FIG.100 and FIG. 101. In some embodiments, the camera can be a Logitech C600Webcamera, the camera sensor can be a ⅓″ 2.0 MP (1600×1200) CMOS:(MI-2010-SOC), the lens can be glass with a standard object distancewebcam lens (Lens-to-Object distance: 35 mm). The light source can be aMoritex White Edge Illuminator MEBL-Cw25 (white) operating at 9.4 volts.Camera images can be taken in a sequence where 1, 2, 3 4, or more tipsare moved by an x-y-z stage into the optical path.

In an embodiment, the detector is a reader assembly housing a detectionassembly for detecting a signal produced by at least one assay on thedevice. The detection assembly may be above the device or at a differentorientation in relation to the device based on, for example, the type ofassay being performed and the detection mechanism being employed. Thedetection assembly can be moved into communication with the assay unitor the assay unit can be moved into communication with the detectionassembly.

The sensors can be PMTs, wide range photo diodes, avalanche photodiodes,single frequency photo diodes, image sensors, CMOS chips, and CCDs. Theillumination sources can be lasers, single color LEDs, broad frequencylight from fluorescent lamps or LEDs, LED arrays, mixtures of red,green, and blue light sources, phosphors activated by an LED,fluorescent tubes, incandescent lights, and arc sources, such as a flashtube.

In many instances, an optical detector is provided and used as thedetection device. Non-limiting examples include a photodiode,photomultiplier tube (PMT), photon counting detector, avalanche photodiode, or charge-coupled device (CCD). In some embodiments a pin diodemay be used. In some embodiments a pin diode can be coupled to anamplifier to create a detection device with a sensitivity comparable toa PMT. Some assays may generate luminescence as described herein. Insome embodiments chemiluminescence is detected. In some embodiments adetection assembly could include a plurality of fiber optic cablesconnected as a bundle to a CCD detector or to a PMT array. The fiberoptic bundle could be constructed of discrete fibers or of many smallfibers fused together to form a solid bundle. Such solid bundles arecommercially available and easily interfaced to CCD detectors.

A detector can also comprise a light source, such as a bulb or lightemitting diode (LED). The light source can illuminate an assay in orderto detect the results. For example, the assay can be a fluorescenceassay or an absorbance assay, as are commonly used with nucleic acidassays. The detector can also comprise optics to deliver the lightsource to the assay, such as a lens or fiber optics.

In some embodiments, the detection system may comprise non-opticaldetectors or sensors for detecting a particular parameter of a subject.Such sensors may include temperature, conductivity, potentiometricsignals, and amperometric signals, for compounds that are oxidized orreduced, for example, O₂, H₂O₂, and I₂, or oxidizable/reducible organiccompounds.

The illumination can be back lit, front lit, and oblique (side) lit.Back lighting can be used in general chemistry for the purpose ofdetecting either light absorption (colorimetry) or scattering(turbidity). The arrangement takes two forms, a broad, evenlyilluminated rear field, and a specifically shaped beam that isinterrupted by the subject. Front lit illumination can be used forreflectance and fluorescence excitation. In reflectance, a subject islit from the front by a light source are measured by observing the lightreflected from the subject. The colors absorbed produce the sameinformation as a liquid illuminated by a back light. In reflectance, asubject can also be illuminated using oblique lighting. The use ofoblique (from the side) illumination gives the image a 3-dimensionalappearance and can highlight otherwise invisible features. A more recenttechnique based on this method is Hoffmann's modulation contrast, asystem found on inverted microscopes for use in cell culture. Obliqueillumination suffers from the same limitations as bright fieldmicroscopy (low contrast of many biological samples; low apparentresolution due to out of focus objects), but may highlight otherwiseinvisible structures.

In fluorescence excitation, subjects can be illuminated from the frontfor the purpose of fluorescence illumination. These are usually singlecolor lights, most commonly lasers. The Confocal Laser ScanningMicroscope is a common embodiment of this. Oblique lighting can also beused in fluorescence excitation. In fluorescence cytometry, the subjectsare often excited at an angle, usually 90 degrees, from which the decayphotons will appear. This form of lighting enables scatter detectiondirectly behind the subject (back lit) as well as the fluorescenceemissions exiting from the side.

In some embodiments, fluorescent light is imaged at 90 degrees to theexcitation beam. In FIG. 102A, a photon source (S), typically ahigh-intensity LED, passes through a beam diffuser (D) and a shapinglens (L1), producing a collimated or slowly diverging excitation beam.The excitation beam passes through a band-pass filter (F1) andilluminates the sample, consisting of a vessel (tube, cuvette, orpipette tip) containing a solution with a fluorescently-labeled sample.Isotropically-emitted fluorescence is spectrally separated fromexcitation light with a long- or band-pass filter (F2) appropriate topass Stokes-shifted fluorescence. Light is then imaged through a lens(L2) onto a digital camera (C) or other detector. Fluorescence intensityis extracted from the resulting images via image analysis.

Images taken using the optical setup shown in FIG. 102A producessingle-tube images (as shown in FIG. 103A. Successive experiments showthe difference in fluorescence intensity from Negative and Positive LAMPexperiments using intercalating dye.

In other embodiments, transmitted light is imaged after opticalfiltering to remove the light at the exciting wavelength. In FIG. 102B,a photon source (S), typically a high-intensity LED, passes through abeam diffuser (D) and a shaping lens (L1), producing slowly divergent,elliptical excitation beam. The excitation beam passes through aband-pass filter (F1) and illuminates the samples, presented as an arrayof sample vessels (tube, cuvette, or pipette tip), each containing asolution with a fluorescently-labeled sample. Isotropically-emittedfluorescence is spectrally separated from excitation light with a long-or band-pass filter (F2) appropriate to pass Stokes-shiftedfluorescence. Light is then imaged through a camera lens (L2) onto adigital camera (C). Fluorescence intensity is extracted from theresulting images via image analysis. The optical setup shown in FIG. 103can be used to produces array images of multiple tubes simultaneously(as shown in FIG. 103B).

For colorimetry, the preferred embodiment for sensing is backlightingthe subject with white light with the result sensed by an imagingsensor. In this case the transmissive color absorption is measured.

For Turbidimetry, the preferred embodiment for sensing is backlightingthe subject with white light with the result sensed by an imagingsensor. For turbidimetry, the reduction of the intensity of thetransmitted light is measured.

Luminometry utilizes no illumination method as the subject emits its ownphotons. The emitted light can be weak and can be detecting using anextremely sensitive sensor such as a photomultiplier tube (PMT).

In some embodiments, imaging may occur using fluorescence, darkfieldillumination, or brightfield illumination. Such imaging can be used forcytometry or other applications. Epi-fluorescence illumination may beachieved by the use of three illumination sources of differingwavelengths. Further, two different sources can be used simultaneously,if required. Consequently, the imaging platform can be used to image alarge variety of fluorescent dyes. The combination of illuminationsources and emission optics can be configured to achieve a plurality ofspectrally independent channels of imaging.

Darkfield illumination may be achieved by the use of a ringlight(located either above or below the sample), a darkfield abbe condenser,a darkfield condenser with a toroidal mirror, an epi-darkfield condenserbuilt within a sleeve around the objective lens, or a combination ofringlight with a stage condenser equipped with a dark stop.Fundamentally, these optical components create a light cone of numericalaperture (NA) greater than the NA of the objective being used. Thechoice of the illumination scheme depends upon a number ofconsiderations such as magnification required, mechanical designconsiderations, size of the imaging sensor etc. A ringlight basedillumination scheme generally provides uniform darkfield illuminationover a wider area while at the same time providing sufficientflexibility in mechanical design of the overall system.

Brightfield illumination may be achieved by the use of a white lightsource along with a stage-condenser to create Koehler illumination.

In some embodiments, an automatic filter wheel may be employed. Theautomatic filter wheel allows control of the imaging optical path toenable imaging of multiple fluorophores on the same field of view.

In some embodiments, image based auto-focusing may take place. Animage-based algorithm may be used to control the z-position (e.g.,vertical position) of an objective (i.e., its distance from the sample)to achieve auto-focusing. Briefly, a small image (for example, 128×128pixels) is captured at a fast rate using darkfield illumination. Thisimage may be analyzed to derive the auto-focus function which is measureof image sharpness. Based on a fast search algorithm the next z-locationof the objective is calculated. The objective may be moved to the newz-location and another small image may be captured. This closed-loopsystem does not require the use of any other hardware for focusing. Themicroscope stage may be connected to computer-controlled stepper motorsto allow translation in the X and Y directions (e.g., horizontaldirections). At every location, the desired number of images is capturedand the stage is moved to the next XY position.

Imaging or other sensing may be performed with the aid of a detector. Adetector can include a camera or other sensing apparatus configured toconvert electromagnetic radiation to an electronic signal. In anexample, a camera can be a charge-coupled (CCD) or electron-multiplyingCCD (EMCCD) camera. A detector may be a sensor, such as an active pixelsensor or CMOS sensor. A detector may include a photo-multiplier tubefor detecting a signal.

The detector can be in optical communication with a sample container(e.g., cuvette, tip, vial). In some cases, the detector is in directline of sight of the sample container. In other cases, the detector isin optical communication with the sample container with the aid of oneor more optics, such as lenses, mirrors, collimators, or combinationsthereof.

Cell counting can be performed using imaging and cytometry. Insituations where the subjects may be bright-field illuminated, thepreferred embodiment is to illuminate the subjects from the front with awhite light and to sense the cells with an imaging sensor. Subsequentdigital processing will count the cells. Where the cells are infrequentor are small, the preferred embodiment is to attach a fluorescentmarker, and then illuminating the subject field with a laser. Confocalscanning imaging is preferred. For flow cytometry, the subjects aremarked with fluorescent markers and flowed past the sensing device.There are two types of sensors, one is position such that the subject isback lit, measuring beam scatter to determine presence of a cell. Theother sensor, aligned so that the illumination is from the side,measures the fluorescent light emitted from the marked subjects. Furtherdescription is provided below relating to imaging methodology forcytometry.

End-User Systems

A device and system may, after manufacturing, be shipped to the enduser, together or individually. The device or system of the inventioncan be packaged with a user manual or instructions for use. In anembodiment, the system of the invention is generic to the type of assaysrun on different devices. Because components of the device can bemodular, a user may only need one system and a variety of devices orassay units or reagent units to run a multitude of assays in apoint-of-care or other distributed testing environment. In this context,a system can be repeatedly used with multiple devices, and it may benecessary to have sensors on both the device and the system to detectsuch changes during shipping, for example. During shipping, pressure ortemperature changes can impact the performance of a number of componentsof the present system, and as such a sensor located on either the deviceor system can relay these changes to, for example, the external deviceso that adjustments can be made during calibration or during dataprocessing on the external device. For example, if the temperature of afluidic device is changed to a certain level during shipping, a sensorlocated on the device could detect this change and convey thisinformation to the system when the device is inserted into the system bythe user. There may be an additional detection device in the system toperform these tasks, or such a device may be incorporated into anothersystem component. In some embodiments information may be wirelesslytransmitted to either the system or the external device, such as apersonal computer or a television. Likewise, a sensor in the system candetect similar changes. In some embodiments, it may be desirable to havea sensor in the shipping packaging as well, either instead of in thesystem components or in addition thereto. For example, adverseconditions that would render an assay cartridge or system invalid thatcan be sensed can include exposure to a temperature higher than themaximum tolerable or breach of the cartridge integrity such thatmoisture penetration.

In an embodiment, the system comprises a communication assembly capableof transmitting and receiving information wirelessly from an externaldevice. Such wireless communication may be Bluetooth or RTM technology.Various communication methods can be utilized, such as a dial-up wiredconnection with a modem, a direct link such as a T1, ISDN, or cableline. In some embodiments, a wireless connection is established usingexemplary wireless networks such as cellular, satellite, or pagernetworks, GPRS, or a local data transport system such as Ethernet ortoken ring over a local area network. In some embodiments theinformation is encrypted before it is transmitted over a wirelessnetwork. In some embodiments the communication assembly may contain awireless infrared communication component for sending and receivinginformation. The system may include integrated graphic cards tofacilitate display of information.

In some embodiments the communication assembly can have a memory orstorage device, for example localized RAM, in which the informationcollected can be stored. A storage device may be required if informationcannot be transmitted at a given time due to, for example, a temporaryinability to wirelessly connect to a network. The information can beassociated with the device identifier in the storage device. In someembodiments the communication assembly can retry sending the storedinformation after a certain amount of time.

In some embodiments an external device communicates with thecommunication assembly within the reader assembly. An external devicecan wirelessly or physically communicate with a system, but can alsocommunicate with a third party, including without limitation a patient,medical personnel, clinicians, laboratory personnel, or others in thehealth care industry.

In some embodiments the system can comprise an external device such as acomputer system, server, or other electronic device capable of storinginformation or processing information. In some embodiments the externaldevice includes one or more computer systems, servers, or otherelectronic devices capable of storing information or processinginformation. In some embodiments an external device may include adatabase of patient information, for example but not limited to, medicalrecords or patient history, clinical trial records, or preclinical trialrecords. An external device can store protocols to be run on a systemwhich can be transmitted to the communication assembly of a system whenit has received an identifier indicating which device has been insertedin the system. In some embodiments a protocol can be dependent on adevice identifier. In some embodiments the external device stores morethan one protocol for each device. In other embodiments patientinformation on the external device includes more than one protocol. Insome instances, the external server stores mathematical algorithms toprocess a photon count sent from a communication assembly and in someembodiments to calculate the analyte concentration in a bodily fluidsample.

In some embodiments, the external device can include one or more serversas are known in the art and commercially available. Such servers canprovide load balancing, task management, and backup capacity in theevent of failure of one or more of the servers or other components ofthe external device, to improve the availability of the server. A servercan also be implemented on a distributed network of storage andprocessor units, as known in the art, wherein the data processingaccording to the present invention reside on workstations such ascomputers, thereby eliminating the need for a server.

A server can includes a database and system processes. A database canreside within the server, or it can reside on another server system thatis accessible to the server. As the information in a database maycontain sensitive information, a security system can be implemented thatprevents unauthorized users from gaining access to the database.

One advantage of some of the features described herein is thatinformation can be transmitted from the external device back to not onlythe reader assembly, but to other parties or other external devices, forexample without limitation, a PDA or cell phone. Such communication canbe accomplished via a wireless network as disclosed herein. In someembodiments a calculated analyte concentration or other patientinformation can be sent to, for example but not limited to, medicalpersonnel or the patient.

Accordingly, the data generated with the use of the subject devices andsystems can be utilized for performing a trend analysis on theconcentration of an analyte in a subject which changes over time.

Another advantage as described herein is that assay results can besubstantially immediately communicated to any third party that maybenefit from obtaining the results. For example, once the analyteconcentration is determined at the external device, it can betransmitted to a patient or medical personnel who may need to takefurther action. The communication step to a third party can be performedwirelessly as described herein, and by transmitting the data to a thirdparty's hand held device, the third party can be notified of the assayresults virtually anytime and anywhere. Thus, in a time-sensitivescenario, a patient may be contacted immediately anywhere if urgentmedical action may be required.

As described elsewhere herein, imaging may be used for detection.Imaging can be used to detect one or more characteristic of a sample.For example, imaging may be used to detect the presence or absence of asample. The imaging may be used to detect the location, placement,volume or concentration of a sample. The imaging may be used to detectthe presence, absence, and/or concentration of one or more analytes inthe sample.

In some embodiments, a single measurement may be used to capture variousinformation about a sample and/or analytes. For example, a singlemeasurement may be used to capture information about the volume of asample and the concentration of an analyte within the sample. A singlemeasurement may be used to capture information about the presence and/orconcentration of a plurality of analytes and/or types of analytes withinthe sample. A single image may be used to capture information relatingto one, two, or more of the information or types of informationdescribed herein.

Such imaging and detection may provide more precise and accurate assays,which may be advantageous in situations with small sample volumes, suchas those described elsewhere herein. Additional examples of volumes ofsample may include 500 μL or less, 250 μL or less, 200 μL or less, 175μL or less, 150 μL or less, 100 μL or less, 80 μL or less, 70 μL orless, 60 μL or less, 50 μL or less, 30 μL or less, 20 μL or less, 15 μLor less, 10 μL or less, 8 μL or less, 5 μL or less, 1 μL or less, 500 nLor less, 300 nL or less, 100 nL or less, 50 nL or less, 10 nL or less, 1nL or less, 500 pL or less, 250 pL or less, 100 pL or less, 50 pL orless, 10 pL or less, 5 pL or less, or 1 pL or less. In some embodiments,the sample volume may include less than or equal to about 3 drops from afingerstick, less than or equal to about 2 drops from a fingerstick, orless than or equal to about 1 drop from a fingerstick. Such smallvolumes may be useful in point of service applications.

Such imaging and/or detection may yield assays with low coefficient ofvariation. A coefficient of variation may be the ratio between thestandard deviation and an absolute value of the mean. In an embodiment,a reaction and/or assay may have a coefficient of variation (CV) (also“relative standard deviation” herein) less than or equal to about 20%,15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, or 0.1%.A single reaction and/or assay, or a procedure with a plurality ofreactions and/or assays may have a coefficient of variation of less thanor equal to about 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.3%, or 0.1%. In some embodiments, an imaging and/ordetection step, or a procedure with a plurality of imaging and/ordetection steps may have a coefficient of variation of less than orequal to about 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5%, 0.3%, or 0.1%.

In some embodiments, the use of imaging with a device that may be placedat a point of service location may improve the overall performance ofthe device. The accuracy and/or precision may be improved and/or thecoefficient of variation may be reduced. The performance of the devicemay be improved when handling small samples, such as those volumesdescribed herein. The imaging may be used in combination with otherdetection systems, in combination with other processes, or as astandalone system. Improvement in performance may include a decrease inthe coefficient of variation of about 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, 0.5%, 0.3%, or 0.1%.

Imaging may be useful for various detection types for one or more typesof assays or sample handling procedures. Examples of such assays orsample handling procedures may include centrifugation, separation,cytometry, immunoassay, ELISA, nucleic acid assay, enzymatic assay,colorimetry, or any other type of assay or reaction described elsewhereherein.

Imaging systems may provide multiple advantages over other methods fordata collection, data processing, and results interpretation. Imagingsystems may maximize, or increase the efficiency of, the use of smallsamples and enhancing system-level performance. Imaging systems may beused for detection as standalone systems or may be used in combinationwith other detection systems or mechanisms.

In some systems, sensors and systems may be used (such as photodiodesand photomultiplier tubes and associated optics/devices) that typicallydo not provide any spatial information about the sample beinginterrogated. Rather, these systems may collect information about thesample after the information has been spatially integrated, typicallylosing spatial information related to the sample. While integrating thesignal in space from the sample may augment the signal levels beingdetected by the sensor, advances in sensitivity of optical and othersensors may negate the need for such integration. Imaging for detectionmay be used in the place of such sensors, or may be used in conjunctionwith such sensors.

Imaging systems may be used that may advantageously have one or more ofthe following features. Imaging sensors may have sensitivity and dynamicrange that meet and/or exceed that of conventional non-imaging sensors.Imaging devices may maintain spatial aspects of the sample beinginterrogated, providing significant ability for post processing. Postprocessing can include QA/QC (e.g., quality control, such as automatederror detection and/or review by pathologist), and/or image analysis toextract specific sample features. The imaging device can utilize 3D, 2D,1D (line sensors), and/or point sensors with a means to translate thesample relative to the collection optics/sensor to enable the spatialreconstruction of the sample. Data collected from the imaging device canbe processed to extract very specific information, such as morphologicalfeatures of the sample (such as cell counts), data from select regionsof the image (peak fluorescence across a sample or in a cell within theimage). Data collected from the imaging device can be processed toimprove the sensitivity and resolution of the measurement. Datacollected from the imaging device can enable the assessment of signalvariation across the sample being imaged. The data may be post processedto calculate mean, standard deviation, maximum, minimum, and/or otherapplicable statistics across the sample or within any regions ofinterest identified in the sample images. Imaging devices enable theexploration of changes in the sample over time by collecting multipleimages and comparing changes in the images over time and space, such aswould be evident in an aggregation processes (such as for an assay ofprothrombin time) or other (e.g., chemical, physical, biologic,electrical, morphological) changes in the sample over time and space.Imaging devices may enable more rapid data acquisition of arrays, tissuesections, and other assay/sample configurations.

Cytometry Application

In some embodiments, any of the embodiments described herein may beadapted to enable the system to perform cytometry. Cytometry (e.g.,enumeration and function analysis of cells) in the system may beperformed using image analysis. Blood can be processed using the pipetteand centrifuge as described previously herein. Typically, a knownmeasured volume of blood (1-50 uL) may first be centrifuged and theplasma fraction removed. The cell fraction may then be re-suspended intobuffer by use of the pipette repeatedly to dispense and aspirate. Acocktail of fluorescent antibodies may be directed to selected cellmarkers (such as CD45, CD4 etc.). Following a brief incubation, areagent which may act as a fixative for the white cells and a lysingagent for red cells can be added. Following another incubation whitecells may be collected by centrifugation and the supernatant hemolysateremoved by aspiration. The stained white cells can be re-suspended in ameasured volume of buffer (typically less than the original blood volume(say 1-20 uL) and dispensed into transparent capillary channels forimage analysis. Typically up to three or even five or more cell typescan be imaged using antibodies having different fluorescent labels orand/or antibodies labeled with different fluor/protein ratios. When morecell types have to be counted or analyzed, more than one reactionmixture can be used. In some embodiments, a reaction mixture can be usedto count or analyze various numbers of cell types.

In some embodiments, the capillary channels are typically about 10-100um deep, 0.5-2 mm wide and 0.5-5 cm long. The capillary channels mayhave other dimensions, including but limited to other dimensionsdescribed elsewhere herein. The stained cell dispersion may fill thechannel usually by capillary action and the cells may be allowed tosettle on the lower channel surface. The channels can be illuminatedwith one or more lasers or other light sources (e.g., LEDs). The opticaltrain may have one or more optical elements, such as dichroic mirrors orlenses, and may or may not magnify the field of view. In someembodiments, the field of view may be magnified 2-100 fold. A series ofimages may be collected typically representing a field of view of about1 mm×0.5 mm and which contains 1-10,000 cells (ideally, 300 cells ofinterest) imaged onto a sensor having an area of about 1000×1000 pixels(1 million total).

A series of images representing adjacent sections of channel may becollected. A mechanical stage can be used to move the channels relativeto the light source. In some cases, a servo-mechanism may move the stagein a vertical direction so as to focus the image. In some embodiments,the light source or one or more optical elements may move relative tothe stage to focus the image. Images are usually made using one or morecombinations of light sources and optical filters. The light sources maybe turned on and off and filters moved into the light path as needed.Preferably up to 1000 cells of any given type may be counted. In otherembodiments, various numbers of cells of any given type may be counted,including but not limited to more than, less than, or equal to about 1cell, 5 cells, 10 cells, 30 cells, 50 cells, 100 cells, 150 cells, 200cells, 300 cells, 500 cells, 700 cells, 1000 cells, 1500 cells, 2000cells, 3000 cells, 5000 cells. Cells may be counted using availablecounting algorithms. Cells can be recognized by their characteristicfluorescence, size and shape. Pattern recognition algorithms may beemployed to exclude stained cell debris and in most cases where thereare cells which are aggregated these can either be excluded from theanalysis or interpreted as aggregates.

A cytometry platform may be an integrated automated microscopy devicecapable of the following tasks in a fully automated, controlledenvironment. One or more of the following tasks may occur in cytometryapplications. The following tasks may occur in the order they appear orin alternate orders or other tasks may be substitute as appropriate.

-   -   1. Isolation of blood cells of the desired type    -   2. Labeling of cells with fluorescent and/or colored dyes and/or        beads    -   3. Confinement of cell suspension in an optically compatible        cuvette    -   4. Imaging of cells using fluorescence microscopy, darkfield        illumination, and/or brightfield illumination    -   5. Automated analysis of images to extract desired cellular        attributes    -   6. Automated analysis of extracted information using advanced        statistical and classification methods to derive clinically        reportable information.

In the following sections, each of these tasks is discussed in greaterdetail; images and sketches are provided wherever deemed necessary.

1. Isolation of Blood Cells of the Desired Type.

Blood cells of a desired type may be isolated in accordance with one ormore embodiments described elsewhere herein. For example, such isolationmay occur as referred to in previous descriptions relating to thecytometry or the centrifuge.

2. Labeling of Cells with Fluorescent and/or Colored Dyes and/or Beads.

Specific fluorescent dyes may be employed. Cells of interest can beincubated with pre-aliquoted solutions of fluorescently labeled binders(e.g., antibodies, aptamers, etc.) which are specific to markers onthese cells. A key consideration may be pairing ‘bright” or highextinction coefficient and high quantum yield fluors with markers forwhich cells have a lower binding capacity; and vice versa. For example,the marker CD22 may be expressed on B-lymphocytes at about one tenth thelevel as CD45. Given this relative expression, CD22 may be labeled witha “bright” dye and CD45 may be labeled with the “dimmer” dye. Themarkers to be labeled using this technique can be either intracellularor cell-surface markers. The sensitivity of detection and quantificationcan be improved by using a secondary labeling scheme for low expressionmarkers. Briefly, a primary binder may be conjugated with anothermolecule which can be specifically recognized by a secondary binder. Asecondary binder labeled with a higher number of fluorophores can thenbind the primary binder in situ and enhance fluorescence signal. Onescheme for achieving this may be the use of biotin conjugated anti-CD22antibody which may be in turn recognized by an anti-biotin antibody thatis labeled with fluorescein isothiocyanate (FITC). The use of candramatically enhance fluorescence signal. FIG. 123 provides an exampleof a fluorescence micrograph showing labeled leukocytes. The exampleillustrates a fluorescence micrograph of Alexa-Fluor 647-anti-CD45labeled human leukocytes in a fixed, lysed blood sample. The pseudocolorscheme is used to enhance perception of the different between ‘bright’cells (with high CD45 expression) and ‘dim’ cells (with low CD45expression).

Color stains of cell smears may also be employed within the system. Forexample, the manual procedure given in StainRITE™ Wright-Giemsa Stain(Polysciences Inc.) can be automated and read in the devices of thesubject invention.

In some embodiments, non-specific fluorescent dyes can be used. For thepurposes of differentiating leukocyte sub-populations, the platform canalso use fluorescent dyes which may bind to nucleic acids (e.g., SYTO,Hoechst) or lipid membranes (e.g., Dil, DiD, FM-4-64).

3. Confinement of Cell Suspension in an Optically Compatible Cuvette.

In some embodiments, cytometry cuvettes may be designed to confine apre-labeled cell suspension of fixed volume into a ‘channel’ fabricatedso as to provide an optically clear imaging material above and below thecells. Sample may be introduced into the channel via a sample entryport. At some distance from the sample entry port, an air vent may allowthe release of air pressure and flow of sample into the channel.

The channel dimensions may be designed to hold a pre-defined knownvolume of fluid, regardless of the volume dispensed at the sample entryport. Each cuvette may have multiple channels of same and/or differentvolumes, each with at least one sample entry port and at least one airvent.

The concentration of cells of interest in the sample can be adjustedduring sample preparation such that after confinement in the cuvette, adesired number of cells per field of view in the imaging system can beachieved. One method for doing this may be to image a container with thecell dispersion and measure turbidity. Using a pre-establishedrelationship between turbidity and cell count, the cell density can becalculated. Typically, the cell dispersion will be made in a volume ofbuffer such that with the lowest likely cell count, and the cellconcentration will be greater than optimal for image-based cellcounting. More buffer may then be added to bring the dispersion to theoptimal level.

The imaging area of the cuvette may be designed so as to provide asufficient number of cells for the application of interest. For example,counting the abundant RBCs may require counting of only 1000-2000 cellsand hence a diluted sample and only a small imaging area in the cuvette.However, counting rare myeloblasts may require in some cases the abilityto image more than 100,000 (total) cells. In such a scenario, the systemmay concentrate the cell suspension so that 100,000 cells may be imagedwith a reasonable number of fields of view. Therefore, the channel onthe cuvette dedicated to RBC imaging will be smaller than the onededicated to imaging myeloblasts.

The cuvette may be designed to be picked up by a standard pipettingmechanism in an automated fashion to allow the transfer of the cuvetteto the imaging platform. The pipetting mechanism's tip ejector can ejectthe cuvette from the pipetting mechanism onto the imaging platform.Registration of cuvette to imaging platform may take place in two steps.Upon transfer of the cuvette to the imaging platform, staticregistration features on the cuvette may interface with mating featureson the imaging platform to align the cuvette parallel to the imagingplatform's optical axis (X,Y registration). Registration may then becompleted by a mechanism located on the imaging platform. This mechanismmay bias the cuvette against a planar surface perpendicular to theimaging platform's optical axis (Z registration), thereby constrainingthe sample within the imaging platform's focal range.

4. Imaging of Cells Using Fluorescence, Darkfield Illumination,Brightfield Illumination.

The method of imaging the cells may also be applied to otherapplications of the invention described elsewhere herein. The imagingtechniques, as previously described, can be used for other imaging uses.

Illumination Capabilities:

The cytometry platform may be designed to have three types ofillumination schemes: epi-fluorescence, darkfield and brightfield. Themodular nature of the setup also allows integration of phase-contrastand differential-interference contrast (DIC).

Epi-fluorescence illumination may be achieved by the use of three laserlines (e.g., 488 nm, 532 nm and 640 nm), but the modular nature of thesystem also allows for integration of other light sources, such as otherlaser sources, LEDs and standard arc-lamps (e.g. Xenon, Mercury andHalogen). Further, two different sources can be used simultaneously, ifrequired. Consequently, the cytometry platform can be used to image alarge variety of fluorescent dyes. The combination of illuminationsources and emission optics can be configured to achieve various numbers(e.g., 3-5) spectrally independent channels of imaging.

Darkfield illumination may be achieved by the use of a ringlight(located either above or below the sample), a darkfield abbe condenser,a darkfield condenser with a toroidal mirror, an epi-darkfield condenserbuilt within a sleeve around the objective lens, or a combination ofringlight with a stage condenser equipped with a dark stop.Fundamentally, these optical components can create a light cone ofnumerical aperture (NA) greater than the NA of the objective being used.The choice of the illumination scheme depends upon a number ofconsiderations such as magnification required, mechanical designconsiderations, or size of the imaging sensor. A ringlight basedillumination scheme generally provides uniform darkfield illuminationover a wider area while at the same time providing sufficientflexibility in mechanical design of the overall system. FIG. 124provides an example of intracellular patterns using darkfield images.The example shows different intracellular patterns in darkfield imagesof human leukocytes. (a) A strong scattering pattern due to presence ofgranules in eosinophils, (b) a polymorphonuclear neutrophil withcharacteristic nucleolar lobes and (c) cells that do not scatter lightto a significant degree (lymphocytes or basophils)

Brightfield illumination may be achieved by the use of a white lightsource along with a stage-condenser to create Koehler illumination. FIG.126 provides an example of brightfield images of human whole blood. Theexample shows brightfield images of a human whole blood smear stainedwith the Wright-Giemsa staining method. Characteristic patterns ofstaining of human leukocytes are apparent. The characteristically shapedred cells can also be identified in these images.

Automatic Filter Wheel:

An automatic filter wheel may allow control of the imaging optical pathto enable imaging of multiple fluorophores on the same field of view.

Image Based Auto-Focusing:

The cytometry platform may use an image-based algorithm to control thez-position (e.g., vertical position) of the objective (i.e., itsdistance from the sample) to achieve auto-focusing. Briefly, a smallimage (for example, 128×128 pixels) may be captured at a fast rate usingdarkfield illumination. This image may be analyzed to derive theauto-focus function which may be used to measure of image sharpness.Based on a fast search algorithm the next z-location of the objectivemay be calculated. The sample may be moved to the new z-location andanother small image may be captured. In some embodiments, thisclosed-loop system does not require the use of any other hardware forfocusing.

Translation of Stage:

The microscope stage may be connected to computer-controlled steppermotors to allow translation in the X and Y directions (e.g., horizontaldirections). At every location, the desired number of images may becaptured and the stage may be moved to the next XY position.

Imaging Sensor:

A camera with a CCD, EMCCD, CMOS or in some cases a photo-multipliertube can be used to detect the signal.

5. Analysis of Images to Extract Desired Cellular Attributes.

The cytometry platform may use different illumination techniques toacquire images that reveal diverse properties and features of the cells.Labeling with cell-marker specific binders may reveal the degree ofexpression of that particular marker on the cell surface or in the cell.Darkfield image may reveal the light scattering properties of the cell.The internal and external features of the cell which scatter more lightappear brighter and the features which scatter lesser amounts of lightappear darker in a darkfield image. Cells such as granulocytes haveinternal granules of size range (100-500 nm) which can scattersignificant amount of light and generally appear brighter in darkfieldimages. Furthermore, the outer boundary of any cell may scatter lightand may appear as a ring of bright light. The diameter of this ring maydirectly give the size of the cell. Brightfield images of cells canreveal cell size, phase-dense material within the cells and coloredfeatures in the cell if the cells have been previously stained.

An image processing library may extract one or more of the followinginformation for each cell (but is not limited to the following):

-   -   1. Cell size    -   2. Quantitative measure of cell granularity (also popularly        called side scatter, based on flow cytometry parlance)    -   3. Quantitative measure of fluorescence in the each spectral        channel of imaging, after compensating for cross-talk between        spectral channels    -   4. Shape of the cell, as quantified by standard and custom shape        attributes such as aspect ratio, Feret diameters, Kurtosis,        moment of inertia, circularity, solidity etc.    -   5. Color, color distribution and shape of the cell, in cases        where the cells have been stained with dyes (not attached to        antibodies or other types of receptor).    -   6. Intracellular patterns of staining or scattering or color        that are defined as quantitative metrics of a biological        feature, for example density of granules within cells in a        darkfield image, or the number and size of nucleolar lobes in a        Giemsa-Wright stained image of polymorphonuclear neutrophils        etc.    -   7. Co-localization of features of the cell revealed in separate        images

The image processing algorithms utilized in this step may usecombinations of image filtering, edge detection, template matching,automatic thresholding, morphological operations and shape analysis ofobjects.

6. Analysis of Extracted Information Using Advanced Statistical andClassification Methods to Derive Clinically Reportable Information.

Any number of measured attributed may be extracted from images of cells.For example, measured attributes of each cell extracted from the imagescan range from 7-15, thus creating a 7 to 15 dimensional space withinwhich each cell is a point. If n measured attributes are extracted fromthe images, an n dimensional space may be provided, within which eachcell is a point.

Based on data acquired for a large number of cells (e.g., 100-100,000cells) a complex n-dimensional scattered data set may be generated.

Statistical methods may be used for clustering cells into individualseparate populations in this n-dimensional space. These methods may alsouse state-of-the-art knowledge from cell biology and hematology to aidin clustering and cell population identification.

FIG. 125 provides an example of multi-parameter acquisition of data fromlabeled cell samples. Human leukocytes were labeled with thepan-leukocyte marker anti-CD45-Alexa Fluor 700 (shown here in green) andthe B-cell marker anti-CD22-APC (shown here in red). The individualchannels show different patterns of CD45, CD22 expression and sidescatter. Cells which are positive for CD22 and CD45 (B-lymphocytes) showthe characteristically low side scatter. On the other hand cells such asneutrophils and eosinophils which have high side scatter do not showlabeling for CD22.

FIG. 127 provides an example of quantitative multi-parametric dataacquisition and analysis. For example, a histrogram may be providedwhich may show distribution of CD45 intensity on human leukocytes. Anyother graphical data distribution techniques may be employed to showsthe distribution. In some embodiments, a scatter plot of side scattermay be provided. The side scatter may be determined by dark-field imageanalysis versus CD45 fluorescence intensity for a human leukocytesample. The side scatter plot may show two main populations ofgranulocytes (top left) and lymphocytes (bottom right).

Foregoing sections describe the main components and capabilities of thecytometry platform and applications. Based on these capabilities, a widegamut of cell-based assays can be designed to work on this platform. Forexample, an assay for performing a 5-part leukocyte differential may beprovided. The reportables in this case may be number of cells permicroliter of blood for the following types of leukocytes: monocytes,lymphocytes, neutrophils, basophils and eosinophils. The basic strategyfor development of this assay on the cytometry platform may be toconvert this into a problem where some attributes of leukocytes aremeasured such as side scatter, CD45 fluorescence intensity, or CD20fluorescence intensity so that leukocytes can be segregated into (e.g.,5) different populations in this n-dimensional space. The regions madearound a cluster of cells can be positioned on a scatter plot in2-dimensional space are called “gates” after flow cytometry parlance. Anexample labeling and “gating” strategy is as follows:

Marker Label Purpose CD2/CRTH2/CD19/CD3 PE-Cy7 Identification oflymphocytes, cocktail labeling of basophils and eosinophils CD45Alexa-Fluor Pan-leukocyte marker to label 647 all leukocytes CD14/CD36cocktail FITC Identification of monocytes

Cell type “Gate” Basophils CD2/CRTH2/CD19/CD3 pos, SSC low, CD45intermediate, CD14/CD36 low Eosinophils CD2/CRTH2/CD19/CD3 pos, SSChigh, CD45 high, CD14/CD36 low Neutrophils CD2/CRTH2/CD19/CD3 neg, SSChigh, CD45 intermediate (less Eosinopils) Lymphocytes CD2/CRTH2/CD19/CD3pos, SSC low, CD45 high, CD14/CD36 low Monocytes CD2/CRTH2/CD19/CD3 neg,SSC int, CD45 int, CD14/CD36 pos

The cytometry platform and analysis system described herein mayadvantageously permit automated sample preparation and execution basedon ordered sample. The systems and methods described may also enablespecific identification of cells as opposed to VCS (volume, conductivityand scatter), which can increase confidence in identification and reduceinstances for confirmatory testing. The image analysis described hereinmay also permit preservation of cell images for later confirmation,analysis as required. There may also advantageously be availability ofmorphological features of the cell. In some embodiments, dynamicadjustment of sample prep and imaging parameters to deal with cellsamples of wide range of concentrations may be provided.

In some embodiments, the centrifuge may be used to prepare andconcentrate cell populations. A method may include the use of thecentrifuge for cell preparation and the imaging and analysis systemdescribed elsewhere herein.

In some embodiments, a combination of dark-field imaging and imaging ofcells stained with multiple fluorescent antibodies may be used. Such acombination may give the equivalent of FACS analysis in a much simplerand less expensive device than other techniques.

In accordance with some embodiments of the invention, the systems andmethods described herein may enable one or more of the followingfeatures. Such features may be advantageous for various applications. Insome embodiments, automated sample inspection and processing may beenabled. Such sample inspection and processing may include one or moreof the following: sample quality, sample volume measurement, dilution(and measurement of dilution factors), and separation of red and whitecells from plasma.

An automated chemical/assay related process may also be employed. Thismay include precipitation, mixing or sedimentation.

In some embodiments, there may be automated measurement of any and allassays that produce luminescence or change light (e.g., colorchemistry). These may include one or more of the following:spectrophotometry, fluorimetry, luminometry, turbidimetry, nephelometry,refractometry, 3-color image analysis, polarimetry, measurement ofagglutination, image analysis (which may employ one or more of thefollowing: camera, digital camera, scanner, lens-less photography, 3-Dphotography, video photography), or microscopy.

Automated quality control and/or calibration of assays may also beprovided within the systems and methods described herein.

In some embodiments, two-way communication may be provided. Suchcommunication may enable record keeping of all assay steps. The two-waycommunication may also enable changes in assay protocols to optimize orincrease completion of multiple assays.

Quality Control/Complementary Applications

In some embodiments, imaging may be used in conjunction with one or moreother measurements or detection steps. The imaging may be complementaryto other techniques, procedures, reactions, and/or assays. For example,imaging may be used to perform one or more quality control check or stepfor any other action, such as a sample preparation, assay, or detectionstep. Imaging may be used for the facilitation of other detections.Imaging may be used to improve the accuracy and/or precision ofcollected data. The imaging may be a quality control aspect to verifydata, results, and/or any measurements. The imaging may be a controlmechanism or improvement mechanism. Imaging may be used to detect one ormore condition that may affect collected data and/or the accuracy and/orprecision of the data. Thus, imaging may improve sample preparation,assay, and/or detection procedures. This may be particularlyadvantageous in situations where there are small sample volumes, such asvolumes described elsewhere herein.

In an example, a detection step may occur to determine the presenceand/or concentration of an analyte. Detection may occur of one or moresignal that may be representative of data that may be useful forsubsequent qualitative and/or quantitative evaluation. Detection may ormay not include the detection of visible light. Detection may includethe measurement of energy from anywhere along the electromagneticspectrum (e.g., infra-red, microwave, ultraviolet, gamma ray, x-ray,visible light). Detection may occur using any type of sensor, which mayinclude an optical sensor, temperature sensor, motion sensor, pressuresensor, electricity sensor, acoustic sensor, chemical sensor,spectrometer, or any other sensor described elsewhere herein, or anycombination thereof. In some embodiments, detection may or may notinclude a spatial distribution of light and/or energy. In someinstances, detection may or may not include an energy densitydistribution.

Imaging may be capable of detecting one or more condition under whichthe detection takes place. Imaging may be used to detect the conditionof a sample, reagent, container, portion of the device that may be usedin the detection. In some embodiments, the imaging may be visibleimaging. For example, imaging may include capturing a snapshot, photo,and/or picture. Imaging may include capturing a spatial distribution ofenergy along the electromagnetic spectrum. The energy along theelectromagnetic spectrum may include visible light, or may include otherranges (e.g., infra-red, ultraviolet, or any other described herein).For example, a spatial distribution of visible light may include atwo-dimensional image. In some embodiments, imaging may include the useof an image capture device, which is described in greater detailelsewhere herein. Some examples of image capture devices may include acamera, such as a lens-less (computational) camera (e.g., Frankencamera)or open-source camera. An image capture device may be capable ofcapturing signals that may be capable of generating a one-dimensional,two-dimensional, or three-dimensional representation of the item that isimaged. In some cases, an image capture device may be a motion-sensinginput device configured to provide a three-dimensional or pseudothree-dimensional representation of an object.

The imaging technique may be the same or may be different from thedetection mechanism utilized. In some instances, different types ofdetection mechanisms are used between the detection step and the qualitycontrol imaging step. In some instances, detection may include an energyband assessment or energy density distribution, such as from aspectrometer, while quality control imaging may include a spatialdistribution of visible light, such as from a camera.

Sensitive detection may be achieved by imaging. For example, an imagingdevice may be able to capture an image to within 1 mm, 500 micrometer(um), 200 um, 100 um, 75 um, 50 um, 25 um, 15 um, 10 um, 7 um, 5 um, 1um, 800 nanometer (nm), 700 nm, 500 nm, 300 nm, 100 nm, 50 nm, 30 nm, 10nm, 5 nm, 1 nm, 500 picometer (pm), 300 μm, or 100 μm. In an example,the imaging may be achieved by a camera which may have a resolution ofgreater than or equal to about 2 megapixels, 4 megapixels, 6 megapixels,8 megapixels, 10 megapixels, 12 megapixels, 15 megapixels, 20megapixels, 25 megapixels, 30 megapixels, 40 megapixels, or 50megapixels, or more.

Imaging may be used to detect an error or other fault state. Imaging maybe used to determine a condition that may increase the likelihood of anerror and/or result in inaccuracies and/or imprecision. For example,imaging may be used to determine the presence and/or absence of one ormore undesirable materials. Examples of undesirable materials mayinclude bubbles, particles, fibers, particulates, debris, precipitates,or other material that may affect a measurement. In another exampleimaging may be used to determine if a volume of sample, reagent, orother material falls within a desired range, or whether a sample,reagent, or other material is located in a desired location. The imagingmay be used to determine the concentration of a sample, reagent or othermaterial, or whether the sample, reagent, or other material falls into adesired concentration range.

In one example, an enzymatic assay may be performed on a small volume ofsample. Examples of volume values may be provided elsewhere herein. Aspectrometer or other detection method or mechanism described herein maybe used to perform a detection step for the enzymatic assay. An imagingstep may occur to determine the conditions under which the detection isoccurring. For example, the imaging step may determine whether there areundesired particulates, such as bubbles, or any other undesiredconditions. The imaging step may verify whether the assay is operatingas it should. The imaging step may confirm whether the operatingconditions under which the assay is occurring and/or detection is beingperformed falls within desired tolerances or optimized conditions. Insome examples, the imaging may include taking a snapshot of a reactionoccurring in a container. The captured image may be analyzed for anyundesirable and/or desirable conditions. In some instances, the capturedimage may be analyzed automatically in a computer assisted method. Oneor more processor may aid with the analysis of the captured image, insome cases using one or more routines implemented by way ofmachine-executable code stored in a memory location. The imaging may beused for quality control without requiring the intervention of a human.

The imaging may provide intelligence for a system. The imaging step mayprovide intelligence on the conditions under which sample preparation,assay, and/or detection occurs. The detection methods may provide morereliable, accurate, and/or precise measurements from a point of servicedevice or component of the device, when utilizing the imaging in aquality control procedure. The quality control may be beneficial whensmall volumes are utilized.

Dynamic Feedback

In some embodiments, dynamic feedback may be provided during a sampleprocessing step. For example, dynamic feedback may occur during a samplepreparation step, assay step, and/or detection step. In someembodiments, dynamic feedback may be provided via imaging.Alternatively, dynamic feedback may occur via any other detectionmechanism, such as those described elsewhere herein. In someembodiments, a dynamic feedback mechanism may utilize optical detection,electromechanics, impedance, electrochemistry, microfluidics, or anyother mechanism or combination thereof.

Dynamic feedback may optionally utilize imaging or other detectionmechanisms. The dynamic feedback may be involved in automated decisionmaking for a system. For example, an image may be captured, and data maybe captured that may be considered in the determination of a step. Asensor, such as an imaging sensor, may capture physical informationwhich may be utilized in the determination of a subsequent step orprocedure. Such subsequent steps or procedures may be determined on thefly in an automated fashion.

In an example, dynamic dilution may occur. A container, such as acuvette or any other container described herein, may have a sampletherein. A dynamic feedback mechanism (e.g., imaging, spectrophotometer,or other detection mechanism) may determine the concentration of asample. In some embodiments, the determination may be a rough or crudedetermination. The initial determination may be a ballpark determinationthat may provide feedback that may put the sample into a condition formore precise or fine-tuned detection and/or analysis. In an example, thedynamic feedback mechanism may be an imaging method that may use aninitial fluorescence detection to do the initial estimate forconcentration.

The dynamic feedback mechanism may determine whether the sampleconcentration falls within an acceptable range. In one example, theconcentration may be a cell concentration. A rough cell count may beperformed to determine cell concentration. One or more signal from thedynamic feedback mechanism may be used for the cell count. In someembodiments, cells may be provided in a wide range of concentrations. Insome instances, the concentrations may vary on over 1, 2, 3, 4, 5, 6, 7or more orders of magnitude. In some embodiments, depending on the cellor analyte to be measured and/or analyzed, different concentrations maybe provided within the same sample. Based on the determinedconcentration, the sample may be diluted or concentrated and/oramplified. For example, if the concentration is higher than a desiredrange, the sample may be diluted. If the concentration is lower than adesired range, the sample may be concentrated and/or amplified. Thedegree of dilution and/or concentration/amplification may be determinedon the fly, based on the estimated concentration.

The degree of dilution and/or concentration/amplification may bedetermined in an automated fashion. Dynamic feedback may be automated.The dynamic feedback mechanism (e.g., imaging or other detectionmechanism) may provide data which may be analyzed to determine anoperational condition. For example, a sample concentration may bedetermined based on the dynamic feedback mechanism. A processor may beprovided, capable of receiving and/or processing one or more signalsfrom the dynamic feedback mechanism. Based on the received signals theprocessor may determine the concentration and whether the concentrationfalls within a desired range. If the concentration falls within thedesired range, the processor may determine that no further dilution orconcentration/amplification is needed. If the concentration is higherthan the desired range, the processor may determine that dilution isneeded. The processor may determine the degree of dilution needed basedon how far the concentration falls outside the desired range. If theconcentration is lower than the desired range, the processor maydetermine that concentration (or amplification) is needed. The processormay determine the degree of amplification needed based on how far theconcentration falls below the desired range. Such determinations may bebased on tangible computer readable media which may include code, logic,or instructions for performing one or more steps. Such determinationsmay be automated and thus made without requiring the intervention of ahuman. This may apply to any operational condition, and need not belimited to sample concentration, such as cell concentration.

In some embodiments, after an initial feedback measurement and dilutionor concentration/amplification step, a more precise measurement may betaken. For example, a more precise measurement of cell counting mayoccur after the sample is determined to be in a desirable range. In someembodiments, a sample may reach a desirable range after a singledilution and/or concentration/amplification step. In other embodiments,additional feedback steps may occur and additional dilution and/orconcentration/amplification steps may be provided, as necessary. Forexample, if an initial determination yields that a sample has a highconcentration, a dilution step may occur. Following the dilution step,an additional feedback step may optionally occur. If the sampleconcentration does not fall into the desired (or otherwisepredetermined) range, an additional dilution orconcentration/amplification step may occur, depending on whether themeasured concentration is above or below the desired range,respectively. This may be repeated as many times as necessary for thesample to fall into the desired range. Alternatively, feedback steps mayor may not be repeated, or may be repeated a fixed number of times. Insome embodiments, each feedback step may occur with a greater degree ofprecision. Alternatively, the same degree of precision may be utilizedin each of the feedback steps.

In some embodiments, when a sample concentration (e.g., cellconcentration, analyte concentration) falls into a desired range, thesample may be analyzed effectively. For example, the sample cellconcentration may have a desired range that may be beneficial forimaging. A desired number of cells per field of view may be provided.

Cell quantification and enumeration by imaging can enhanced bycontrolling the cells density during imaging, thus limiting crowding andclustering of cells. Consequently the range of analyte concentrationover which the assay is linear can be maximized or increased. In orderto extend the assay linear range, the dynamic system may perform aprior, non-destructive measurement on the sample using a method whichhas a high dynamic range to provide determine a rough cell concentrationin the sample. An algorithm may then calculate the dilution ratiorequired to bring the cell concentration in the acceptable range for themain measurement. Dilution and/or concentration/amplification may beprovided accordingly, thereby providing dynamic dilution and/orconcentration.

Such dynamic feedback, such as dynamic dilution, may be advantageous insystems utilizing small volumes. In some embodiments, a total samplevolume may include any of the volumes described elsewhere herein. Insome instances, the volumes for a particular portion of a sample to beanalyzed may have any of the volumes described elsewhere herein. Dynamicdilution may assist with providing low coefficient of variation. Forexample, a coefficient of variation for a sample preparation, assay,and/or detection step may have a coefficient of variation value asdescribed elsewhere herein. This may be advantageous in point of servicedevices, which may utilize small volumes, and/or have low coefficientsof variation.

Dynamic feedback may advantageously permit non-destructive testing of asample. This may be advantageous in systems using small volumes. Thesame sample may be used for the initial feedback detection and forsubsequent detections. The same sample may under initial feedbackdetection and subsequent detections within the same container (e.g.,cuvette, vial, tip). A vessel may be provided with a sample that isoutside a desired and/or detectable range in its initial state. Forexample, a concentration of one or more analytes and/or cells may falloutside a desired and/or detectable concentration range initially. Thesame sample may be measured within the range in the same vessel. In someembodiments, the concentration of the one or more analytes and/or cellsmay later fall within a desired and/or detectable range in the samevessel. In some embodiments, one or more intervening steps, such asdilution and/or concentration/amplification may be performed on thesample in order to get the sample into the desired and/or detectablerange. Such intervening steps may be performed in an automated fashion.

In some embodiments, dilution may be provided to the sample in anautomated fashion. For example, a diluent may be dispensed into acontainer holding the sample and mixed with the sample to effect a newsample volume. In some cases, the diluent includes a single diluent. Inother cases, the diluent includes a plurality of diluents. The diluentcan be dispensed into the container with the aid of a pumping system,valves and/or fluid flow channels for facilitating the flow, such as amicrofluidic system having one or more microfluidic channels and/or oneor more microfluidic pumps. The microfluidic system may include one ormore mechanical and/or electromechanical components, such as amechanical pumping system having one or more actuated (e.g.,pneumatically actuated) valves for facilitating the flow of a fluid. Thepumping system in some cases includes a mechanical pump configured tofacilitate fluid flow. The pumping system can include one or moresensors for measuring and relaying operating parameters, such as fluidflow rate, concentration, temperature and/or pressure, to a controlsystem. In an example, the diluent is dispensed into the container withthe aid of a microfluidic system having a mechanical pump coupled to amicrofluidic channel bringing the container in fluid communication witha diluent reservoir.

In some cases, a pumping system is provided to release a diluent basedon a measured sample dilution. The sample dilution can be measured withthe aid of a sensor, such as, for example, a light sensor. In anexample, the light sensor is coupled with a light source for directing abeam of light through the sample, and subsequently measuring sampledilution based at least in part on the scattering of light through thesample. If the measured sample (e.g., cell, tissue) concentration isabove a predetermined limit (or threshold), then the pumping systemdirects a diluent (e.g., water) from a diluent reservoir to a containerholding the sample.

In some embodiments, dynamic dilution is electronically automated withthe aid of a fluid flow system having a pump (e.g., microfluidic pump)in fluid communication with a fluid flow channel (e.g., microfluidicchannel), and further including one or more valves for regulating fluidflow. The automation of dilution can be used to test and/or adjustcalibration settings, such as preset dilution fluid volumes used toeffect a desired concentration.

In some situations, the pump comprises one or more valves, such aspneumatically-actuated valves. The pump, fluid flow channel and one ormore valves bring a diluent reservoir in fluid communication with acontainer configured to hold a sample. The one or more valves and/or thepump can be in electrical communication with a control system having aprocessor for regulating the flow of diluent from the diluent reservoirto the to regulate the concentration of the sample.

Dynamic feedback advantageously enables the automated regulation ofsample concentration while minimizing, if not eliminating, userinvolvement. In some cases, the concentration of a sample isautomatically regulated (e.g., diluted or amplified) without any userinvolvement. Such minimal user involvement can provide low coefficientof variation in imaging and overall system use, as described elsewhereherein.

In an example, dynamic feedback system is used to regulate theconcentration of cells in a fluid sample using imaging. With the sampleprovided in a sample container, such as cuvette, the imaging is used tomeasure the concentration of cells in the fluid sample. The measuredconcentration can be a rough (or ballpark) measurement of concentration.The dynamic feedback system then dilutes the fluid sample by providing adiluent into the sample container. This may minimize, if not eliminate,any disturbance to (or destruction of) the cells upon dilution. Anoptional measurement of the concentration of cells in the fluid samplecan then be made to measure the concentration following dilution. Insome situations, following dilution a reaction can take place in thesame sample container that was used to dilute the sample. In somesituations, the reaction may take place in cases in which the dilutionis not optimal.

In some cases, during dynamic feedback a rough measurement of sampleconcentration is made with the aid of a spectrometer, and a more precisemeasurement of sample concentration is made with the aid of an imagingdevice. The imaging device can include a light source (e.g., coherentlight, such as a laser, or incoherent light) and a camera, such as acharge-coupled device (CCD) camera. In an example, following the roughmeasurement, the dynamic feedback system coarse adjusts theconcentration of the sample by providing the diluent, and subsequentlymakes the more precise measurement. The sample concentration can befurther adjusted by providing smaller volumes of a diluent (i.e., fineadjustment) in relation to the volume of the diluent provided duringcoarse adjustment. Alternatively, the rough measurement of sampleconcentration is made with the aid of an imaging device, and the moreprecise measurement is made with the aid of a spectrometer. Coarse andfine adjustment

Dynamic feedback systems provided herein can be configured toconcentrate/amplify (i.e., increase the concentration of) a sample, suchas cells in a fluid sample. In some cases, this is accomplished with theaid of centrifugation or field-induced separation (e.g., electric fieldseparation, magnetic separation).

In some situations, the concentration of a sample is made using animaging device, with the location of the imaging device selected toselect a desired path length and/or focal point. In some cases, thelocation of one or more optics associated with the imaging device areadjusted to provide a desired path length and/or focal point. In somecases, a lens-less camera is used for image capture, which cancomputationally provide image analysis and various focal points.

Dynamic dilution can be performed on various sample volumes. In somecases, if a sample volume is above a predetermined limit, the sample canbe distributed in multiple sample containers (e.g., cuvettes) forsequential or parallel processing and/or imaging.

Self-Learning

The dynamic feedback mechanism may result in self-learning by thesystem. For example, for a dynamic dilution/concentration system, aninitial feedback measurement may be made. Based on the feedbackmeasurement, the sample may have no action, may be diluted, or may beconcentrated/amplified. Subsequent measurements and/or detection mayoccur. The subsequent measurements and/or detection may or may not beadditional feedback measurements. Based on the subsequent measurements,a determination may be made whether the action taken (e.g., no action,dilution, concentration/amplification) was correct and/or whether thecorrect degree of action was taken (e.g., enough dilution orconcentration/amplification). For example, an initial feedback mechanismmay determine that the sample concentration is high and needs to bediluted. The sample may be diluted by a particular amount. A subsequentmeasurement may be taken (e.g., image of the sample may be taken). Ifthe degree of dilution does not bring the sample into the desired range(e.g., dilution was too much or too little), the system may receive anindication that for subsequent dynamic dilutions/concentrations with thesame or similar initial feedback mechanisms, a different degree ofdilution may be used. If the degree of dilution does bring the sampleinto the desired range, the system may receive a confirmation that theamount of dilution should be used for subsequent dilutions for the sameor similar type of initial feedback measurement.

Data points may be gathered based on initial conditions and subsequentactions, which may assist with determining appropriate actions to takein subsequent dynamic feedback situations. This may cause the system toself-learn over time on steps to take in particular dynamic situations.The self-learning may apply to individualized situations. For example,the self-learning system may learn that a particular individual fromwhom the sample is drawn, may require different degrees ofdilution/concentration than another individual. The self-learning mayapply to groups of individuals having one or more characteristic. Forexample, the self-learning system may learn that an individual using aparticular type of drug may require different degrees ofdilution/concentration than another individual. The self-learning systemmay also be generalized. For example, the system may become aware of apattern that people of a particular demographic or having particularcharacteristics may or may not required different degrees of dilutionand/or concentration. The system may draw on past data points,individuals' records, other individuals' records, general healthinformation, public information, medical data and statistics, insuranceinformation, or other information. Some of the information may bepublicly available on the Internet (e.g., web sites, articles, journals,databases, medical statistics). The system may optionally crawl websites or databases for updates to information. In some embodiments,self-learning may occur on the device, the cloud or an external device.As additional data is gathered, it may be uploaded to the cloud orexternal device, and may be accessible by the self-learning system.

Image Capture and/or Manipulation Devices

In some embodiments, sample preparation, processing and/or analysis isperformed with the aid of image capture and/or manipulation devices,including electromagnetic radiation (or light) capture and/ormanipulation devices, such as imaging devices or spectrometers. In somecases, an imaging device can be used in association with a spectrometer.A spectrometer can be used to measure properties of light over a selectportion of the electromagnetic spectrum, which may be used forspectroscopic analysis, such as materials analysis. An imaging (or imagecapture) device can be used to measure sample concentration,composition, temperature, turbidity, flow rate, and/or viscosity.

In an example, an image capture device may be a digital camera. Imagecapture devices may also include charge coupled devices (CCDs) orphotomultipliers and phototubes, or photodetector or other detectiondevice such as a scanning microscope, whether back-lit or forward-lit.In some instances, cameras may use CCDs, CMOS, may be lens-less(computational) cameras (e.g., Frankencamera), open-source cameras, ormay use any other visual detection technology known in the art. In someinstances, an imaging device may include an optical element that may bea lens. For example, the optical element is a lens which captures lightfrom the focal plane of a lens on the detector. Cameras may include oneor more optical elements that may focus light during use, or may captureimages that can be later focused. In some embodiments, imaging devicesmay employ 2-d imaging, 3-d imaging, and/or 4-d imaging (incorporatingchanges over time). Imaging devices may capture static images or dynamicimages (e.g., video). The static images may be captured at one or morepoints in time. The imaging devices may also capture video and/ordynamic images. The video images may be captured continuously over oneor more periods of time.

In some cases, an image capture device is a computational camera that isused to measure the concentration of a plurality of samples within arelatively short period of time, such as at once. In some embodiments,the computational camera may have an optical which may be different froma lens. In an example, the computation cameral is a lens-less camerathat takes a photograph of a plurality of samples in staggered samplecontainers (e.g., cuvettes). The concentration of a sample in aparticular sample container can then be calculated by, for example,mathematically rendering the image to select a focal point at oradjacent to a portion of the image having the particular samplecontainer, and deriving the sample concentration from the renderedimage. Such mathematical manipulation of an image, as may be acquiredwith the aid of a lens-less camera, can provide other information atvarious points in space within the field of view of the lens-lesscamera, which may include points in space that may be extrapolated fromscattered light. In some embodiments, the final signal may be analyzedby complex algorithms. One example of such a setup is a computationalcamera with optical elements which may produce a Fourier-transformedimage on the detector. The resulting “image” can be analyzed to extractrequired information. Such a detector would enable one to obtain richinformation from the imaged subject. Obtaining different features fromthe image, for example, information at a different focal length could bedone purely through software, simplifying the imaging hardware andproviding more rapid and informative data acquisition.

Electromagnetic radiation capture and/or manipulation devices can beused in various applications provided herein, such as measuring sampleconcentration, including dynamic dilution. In an example, a lightcapture and/or manipulation device includes a light source, such as acoherent light source (e.g., laser), coupled with a light sensor, suchas a CCD camera, for capturing scattered light, as may emanate from asample upon the light source being directed through the sample. This canbe used to measure the concentration of the sample. The light sensor canbe configured to capture (or sense) various wavelengths of light, suchas red, green and blue, or other color combinations, such ascombinations of red, orange, yellow, green, blue, indigo and violet, toname a few examples. In some situations, the light sensor is configuredto sense light having wavelengths at or greater than infrared or nearinfrared, or less than or equal to ultraviolet, in addition to thevisible spectrum of light.

Light capture and/or manipulation devices can be used to collectinformation at particular points in time, or at various points in time,which may be used to construct videos having a plurality of still imagesand/or sound (or other data, such as textual data) associated with theimages.

Light capture and/or manipulation devices, including computational (orlens-less) cameras, can be used to capture two-dimensional images orthree-dimensional (or pseudo three-dimensional) images and/or video.

In some embodiments, an image capture and/or manipulation deviceperturbs an object and measures a response in view of the perturbation.The perturbation can be by way of light (e.g., x-rays, ultravioletlight), sound, an electromagnetic field, an electrostatic field, orcombinations thereof. For example, perturbation by sound can be used inacoustic imaging. Acoustic imaging may use similar principles todiagnostic ultrasound used in medicine. Acoustic imaging may functionsimilarly to a regular microscope but may use acoustic waves. A sourceof ultrasound may produce waves that can travel through the sample andget reflected/scattered due to heterogeneities in the elastic propertiesof the sample. The reflected waves may be “imaged” by a sensor. Avariant of this method may include “photo-acoustic imaging” where theacoustic waves traveling through the sample may cause local compressionand extension of the sample. This compression/extension may cause achange in the refractive index of the sample material which can bedetected by measuring/imaging the reflection of a laser beam by thesample.

In some situations, an imaging device can be used to measuring thevolume of a cell. In an example, the combination of a light source andCCD camera is used to capture a still image of a cell. A computer systemdigitizes the still image and draws a line across the cell, such asthrough the center of the cell. The computer system then measures thedistance between the points at which the line intersects the boundariesof the cell (or cell wall or membrane) to provide an estimate of thediameter of the cell, which may be used to estimate the volume of thecell.

The imaging device may utilize line scanning microscopy to enable sampleillumination with a thin line or spot of coherent laser light, so thatpower from the source can be concentrated in a small area giving highpower densities. The detector geometry may be matched with the line orspot. Then the line/spot may be scanned across the sample so thatdifferent parts of it can be imaged. Each scanned line can then beconcatenated to form the whole image (e.g., in a similar manner like adocument scanner). This method may be advantageous as ananalytical/imaging method for one or more of the following reasons: (1)high power density of illumination, (2) relatively high speeds can beobtained from line scanning as opposed to spot scanning, (though bothmay be slower than full-frame or classical imaging), (3) high precisionand/or accuracy of analytical measurements on the sample such asfluorescence, absorbance, luminescence, (4) combination with spectral orhyper-spectral imaging such that a complete spectrum of the sample canbe acquired for each pixel, (5) on-the-fly adjustment of resolution,(i.e. without changing any elements, a sample can be scanned at low orhigh lateral resolution as desired), or (6) can provide high depth offield to allow imaging of tissue samples.

In some embodiments, an imaging device is configured to detect lightemanating from an ionization (fluorescence or luminescence) event, suchas via scintillation. In an example, a scintillator is coated on orembedded in a material comprising a sample container. Upon a samplebinding to (or otherwise interacting with the scintillator), thescintillator emanates light (e.g., fluorescent light) that is detectedby a detector of the imaging device. This may be used to measure theradioactive decay (e.g., alpha and/or beta decay) of certain samples.

In some situations, an imaging device is a field effect transistor fordetecting charged particles, such as ions. Alternatively, the imagingdevice may be a thermal detector for measuring a heat change, which maybe used to construct a heat map, for example.

In some situations, a sample container comprises one or more wells forimmobilizing a sample. The sample container may be coupled with animaging device for imaging a sample immobilized in the one or morewells. Sample immobilization can be facilitated with the aid of beadshaving surface binding agents (e.g., antibodies) or surface bindingagents, which may be disposed, for example, at bottom portions of wells.The wells can have diameters on the order of nanometers to micrometersor greater.

In some embodiments, sample detection and/or analysis is facilitatedwith the aid of image enhancement species, such as dyes. A dye may bindto a sample provide an optical, electrical or optoelectronic signal thatcan be detected by a detector of an imaging device. In an example, a dyebinds to a cell and fluoresces, which is recorded by a detector. Bymeasuring fluorescence, the spatial distribution and/or concentration ofcells can be measured. Image enhancement species can aid in achievingimproved signal-to-noise during image acquisition (or capture). A dyecan bind to a cell with the aid of surface receptors and/or antibodies.

In some cases, the use of dyes can generate background fluorescence,which may distort an image—the fluorescence sample may be difficult toresolve from the fluorescing background. In such a case, imageacquisition can be enhanced by contacting a sample in a fluid with afluorescent dye. Unbound dye is removed with the aid of a centrifuge (ormagnetic or electric separation), which separates the sample from theunbound dye. The centrifuge may be integrated in a point of servicedevice having the imaging device. The sample can then be re-suspended ina fluid and subsequently imaged with the aid of the imaging device.

In some cases, image acquisition can be enhanced by using dynamicfeedback in addition to, or in place of, the use of image enhancementspecies. In an example, the concentration of the sample is optimizedwith the aid of dilution and/or amplification prior to imageacquisition.

Sample separation can be facilitated with the aid of a centrifuge. As analternative, sample separation can be performed with the aid of amagnetic or electric field. For instance, a magnetic particle can bindto a cell, which in the presence of a magnetic field can be used toattract the cell towards the source of magnetic attraction.

Systems and methods provided herein can be applied to various types ofsamples, such as cells as may be derived from tissue (e.g., skin,blood), saliva or urine. In an example, dynamic feedback and/or imagingcan be applied to tissue samples or cell samples derived from suchtissue samples.

EXAMPLES Example 1: Nucleic Acid Amplification by Loop-MediatedIsothermal Amplification (LAMP)

To evaluate the ability of the three-color image analysis method forboth fluorescence and absorption to read LAMP assays the followingexperiments were performed.

Lamp Reaction Conditions

The LAMP reaction was carried out in a total volume of 25 μL in 500 uLPCR tubes (VWR, West Chester, Pa.). The reaction mixture included 0.8 μMof primer 1 and primer 2, 0.2 μM of primer 3 and primer 4, 400 μM eachdNTP (Invitrogen, Carlsbad, Calif.), 1M betaine (Sigma, St. Louis, Mo.),1× Thermopol Buffer (New England Biolabs, Ipswitch, Mass.), 2 mM MgSO4(Rockland Immunochemicals, Gilbertsville, Pa.), 8U Bst DNA polymeraselarge fragment (New England Biolabs, Ipswitch, Mass.), and a givenamount of template DNA (varied between ˜10 and ˜10{circumflex over ( )}9copies). In the case of negative control approximately 10{circumflexover ( )}9 copies of irrelevant DNA was added.

Reaction Conditions

The reaction was incubated at 65° C. for 1 hour in sealed tubes. Thepolymerase was then inactivated by heating the reaction product to 80°C. for 5 minutes.

Product Detection and Visualization

SYBR Green I stain (Invitrogen, Carlsbad, Calif.) stock was diluted 100fold, 5 μL was mixed with 10 μL of the completed LAMP reaction productmixed, and incubated for 5 minutes at room temperature. The reactionproducts were then read out in the following way:

Fluorescence readout: PCR tubes or pipette tips containing the mixture,were illuminated with 302 nm UV light and fluorescent emission (λmax˜524nm) imaged by a digital camera (Canon EOS T1i, 18-55 mm, Canon, LakeSuccess, N.Y.).

Color readout: Reaction products were aspirated into tips and imagedusing a digital camera

Results:

A fluorescence image of assay products in tubes is shown in FIG. 81.

A fluorescence image of the assay product in tips is shown in FIG. 89.

Color images of the assay products in tips are shown in FIG. 82, FIG.83, FIG. 84, FIG. 85, FIG. 86, and FIG. 87. FIG. 88 shows a backgroundcolor image obtained for calibration.

FIG. 90 shows a comparison of LAMP dose-responses obtained bymeasurement of “bulk” fluorescence (conventional fluorometry) andresponses for two color channels obtained by camera. As is evident, thecolor method shows a response comparable to that of fluorimetry.

When the color images were analyzed and calibrated according to methodsdescribed herein using all thee color channels, the close correspondenceof the calibrated color signal with the fluorescence signal is evidentas shown in FIG. 91.

Example 2: System Maximizing Sample Utilization

A system for maximizing sample utilization can have the followingcharacteristics:

1. Efficient separation of blood into plasma and efficient recovery ofthe plasma

-   -   a. Separation is achieved by centrifugation in a capillary tube

2. Dilution of the plasma to a few pre-established levels appropriate toboth high and low sensitivity assays

3. Minimizing the volume of each assay reaction mixture required foreach assay

-   -   a. Using an open-ended low volume cuvette suitable for assay        incubations while precluding evaporation        -   i. Cuvette is long relative to width    -   b. Within said low volume cuvettes enabling increase in assay        signal sensitivity by modifying the optical pathlength        -   i. Cuvette is conical or has features where the width is            wide and narrow    -   c. When needed, achieving said increase in assay signal        sensitivity by moving the reaction product (which does not fill        the cuvette) to selected locations having greater pathlength at        the time of optical measurement        -   i. Cuvette internal volume is much larger than the volume of            the assay mixture

4. Use of either or both variable pathlength and 3-color channelanalysis to increase the useful dynamic range of assays

Example 3: Point-of-Care Assay Device

A point of care assay device can include of single-use disposablecartridges an instrument which processes samples and operates the assaysand a server remote from the instrument, the measurement and detectionsystem comprising:

-   -   A disposable cartridge containing        -   Sample-acquisition and metering methods (such as a sample            tip)    -   An instrument housing containing        -   A light imaging sensor (such as a cell-phone camera having a            light source (e.g., a flash) and a CCD image collecting            device)        -   A mechanism for moving said tip to a location where said            light imaging sensor can acquire images    -   Uploading said images wirelessly (or by other methods) to a        server remote from the instrument    -   Image interpreting software capable of:        -   Measuring volumes from the two-dimensional images        -   Distinguishing sample types    -   Using said sample type and/or volume data as part of an        operating algorithm to:        -   Provide prompts to system users as to sample integrity        -   Provide any needed prompts to provide an augmented or            replacement sample        -   Interpret signal data from said instrument in terms of assay            results making allowance for sample type and/or sample            volume

The system can optionally include additional mechanisms for processingand/or imaging of samples acquired by users into a “sample acquisitiondevice (capillary)” comprising:

-   -   Mechanisms for accepting the capillary in a defined location and        moving said capillary to another defined location where an image        can be acquired    -   Mechanisms for ejecting substantially all the sample into the        said cartridge at a defined location

Example 4: Analysis of a Capillary Containing a Blood Sample

Samples are acquired by users by touching the distal end of thecapillary to a drop of blood (typically as a fingerstick). The capillaryusually fills by capillary action provided there is sufficient blood. Inone version of the invention, the user places the capillary at alatching location on the cartridge then inserts the cartridge onto aslide in the instrument then activates an assay by pressing a screenbutton on the instrument GUI. All subsequent assay steps are automated.The instrument moves the cartridge inside its housing and closes thedoor through which the cartridge was inserted. In this version of theinvention, the instrument moves a component for grasping the capillaryand moving it to a location in front of a digital camera equipped with aflash light source a CCD. An image of the capillary is taken using flashillumination and sent wirelessly to the server which interprets theimage in terms of type, location and quantity of sample. If presetcriteria are met the server instructs the instrument to continue theassay. If the sample is not appropriate the capillary is returned to thecartridge which is ejected and the server causes the GUI to display anappropriate prompt. The user may then (1) add more sample or (2) obtaina fresh sample and use a new capillary. Once the user indicates by theGUI that corrective action has been taken and the capillary/cartridgehas been re-inserted into the instrument, the server instructs theinstrument to resume assay processing. The criterion for appropriatevolume of sample is usually that the volume is more than the minimumrequired for the assay. Thus in some assays for example, 10 uL of samplecan be used, so typically the sample is regarded as adequate if themeasured volume is >12 uL.

In a second version of the invention which may be implemented alone orin combination with the first, image acquisition is used to measure thevolume of sample taken from the original sample by the instrument. Inthe assay sequence, sample is ejected from the capillary into a samplewell in the cartridge either (1) by the user, or (2) by the instrument.Then an exact volume is taken from sample well using a second tip eitherby capillary action or (preferred) pneumatic methods. At this stage thetype of the sub-sample and the sub-sample volume is measured (as above)by imaging the tip. If the sample type and volume is acceptable(target+/−<5%), the assay proceeds. If not, the assay may be aborted andthe user prompted to take remedial action. Sample types that may bediscriminated are blood and plasma or serum and others. The imagingsystem makes the distinction by observing the much greater contrastbetween blood (opaque) and the tip (transparent) than is the case forplasma and serum. In the event that the sample volume while not at thetarget level is still sufficient for the assay to give satisfactoryresults (in the above example, a volume >5 uL would be acceptable if thetarget volume is 10 uL). The assay algorithm that calculates the analyteconcentration then uses a correction function Conc. (true)=Conc.(observed assuming target volume)*Target volume/Measured volume.

Blood can easily be detected and its volume measured by creating a pixelmap of the tip and counting the dark pixels then comparing with apreviously established number for the target volume. Even though sampletypes serum and plasma (and other aqueous non-blood samples) aretransparent, the imaging system can still detect the presence of sampledue to the change in refraction that occurs over the sample meniscus andthe difference in refractive index between the tip material and thesample. Alternatively a dye may be added to the sample by providing adried dye formulation coated within the capillary which is dissolved bythe sample

Other methods for measuring the volume of sample include locating thetop and bottom of each meniscus and using simple geometric techniques(as described herein). Bubbles within the sample liquid column can berecognized and measured by the methods discussed above and theappropriate volume subtracted from the total volume occluded by thesample.

The methods given above measure the sample within the sample capillaryor pendent from the end of the capillary (as described herein). Afterthe sample is measured and accepted by the system it is ejected bypipetting/pneumatic methods within the instrument. Once this hashappened, the tip can be imaged again and any residual sample measured.The volume that actually is used in the assay is the difference betweenthe total volume and the residual volume.

Another particular problem in POC assay systems especially when used bynon-technically trained users is the presence of sample on the outsideof the sample capillary.

This can be imaged and measured using the invention and the userprompted to remove excess blood.

The effectiveness of sample acquisition and delivery in assay devicesdepends on the liquid handling techniques used. Automated devices mayuse (1) pneumatic aspiration and ejection (as in many laboratory singleand multi-channel pipetting devices that use disposable tips; pneumaticmethods may use positive or negative pressure (vacuum)), (2) positivedisplacement (as in a syringe), ink jet-like technology and the like.Samples and other liquids such as reagents can be (3) drawn out ofreservoirs by capillary action or (4) wicking into porous media. Liquids(samples and/or reagents) may be ejected with or without contactingother liquids. For example, if the sample is to be diluted, the sampletip can be dipped into the diluent or displaced into air so as to dropinto a dry well or a well containing diluent. The performance of all ofthe above systems and methods may be verified and/or measured using theinvention.

In other embodiments, the capillary can be imaged by a user outside theinstrument with an external camera. Volume measurements can be scaled tothe size of the capillary. Such an externally oriented camera can beused for recognition of the user/patient so that results can morereliably be attributed to the correct patient. The method may also beused to verify appropriate medication is being used (image the pillcontainer or pill or alternatively the bar code reader in the instrumentmay be used for this purpose).

The invention may also be used to measure location and volumes ofreagent aspirated into assay tips. In some cases dyes may be added toreagents to make them more easily imaged (improved contrast).

In assays where plasma is separated from blood, the invention can beused to verify the effectiveness of red cell removal and the availablevolume of plasma. An alternative to moving the sample containing tip isto move the camera

Such a system can have the following advantages:

1. Quantitative measurement of sample

2. Ability to identify the sample type

3. Creation of an objective, quantitative record of sample volume

4. Enables assays to give results when sample volume is not correct

5. Improves reliability of assay system

Example 5: Tips

FIG. 18 shows diagrams of tips used to aspirate samples and reagents(dimensions in mm).

Example 6: Geometry Measurements of a Cylindrical Capillary

FIG. 19 shows dimensions of a cylindrical capillary containing a sample.

R=radius

L1=distance from lower end of cylinder to lower sample meniscus

L2=distance from lower end of to lower upper meniscus

Volume introduced=π*(R{circumflex over ( )}2)*(L2−L1)

Example 7: Geometry Measurements of a Conical Capillary

FIG. 20 and FIG. 21 shows dimensions of a conical capillary.

Rb=radius at base of cone

L=length

L₁=distance from (projected) top of the cone to lower sample meniscus

L₂=distance from (projected) top of the cone to lower upper meniscus

Volume introduced=π*(Rb/L){circumflex over ( )}2*[(L ₁){circumflex over( )}3−(L ₂){circumflex over ( )}3]/3

Tan θ=Rb/L

Example 8: Effects of Liquid Meniscus

As is well known, liquids in capillaries typically have a curvedmeniscus. Depending on the contact angle the meniscus may be curvedinward or outward relative to the liquid. When no net external pressureis applied, if the capillary surface is hydrophilic (contact angle <π/2)the meniscus is inward directed or outward directed if the surface ishydrophobic (contact angle >π/2). When net pressure is applied to theliquid column (capillary oriented vertically or pneumatic pressureapplied by the instrument) the lower meniscus can project below thelower end of the capillary. In small diameter capillaries, surfacetension forces are strong relative to the small gravitational forceacross a meniscus. Surface tension pressure across a meniscus in avertically oriented circular capillary is 2π*R*γ*cos θ where γ is thesurface tension and θ is the contact angle. Pressure across a meniscuscaused by gravity is ρgΔL/(π*R{circumflex over ( )}2) where p is liquiddensity and ΔL is the distance across the meniscus and g is thegravitational constant. Accordingly the meniscus surface is spherical.The volume of liquid in the segment(s) occupied by the meniscus(menisci) can be calculated as follows and used to obtain a moreaccurate estimate of volume.

Distances defined from the bottom of the sample capillary

L1=distance to the bottom of the lower meniscus

L2=distance to the top of the lower meniscus

L3=distance to the bottom of the upper meniscus

L4=distance to the top of the upper meniscus

Volume of a spherical cap

πh(3a ² +h ²)/6

Dimensions of a spherical cap are shown in FIG. 22.

Several different situations can arise defined by the number andlocation of the menisci. Note that the formulae given below deal withboth inward and outwardly curved menisci.

Case 1: Upper meniscus is curved, lower is horizontal (as shown in FIG.23)

Substituting: a=R, h=L₄−L₃

Volume between L₃ and L₄=π*(L₄−L₃)*(3*(R)²+(L₄−L₃)²)/6

Total volume=π*((R ²)*L ₃+(L ₄ −L ₃)*(3*(R)²+(L ₄ −L ₃)²)/6)

Case 2: Both menisci are within the capillary and curved (as shown inFIG. 24)

Total volume=π*((R ²)*L ₃−(L ₂ −L ₁)*(3*(R)²+(L ₂ −L ₁)²)/6+(L ₄ −L₃)*(3*(R)²+(L ₄ −L ₃)²)/6

Case 3: There are two curved menisci. The lower being curved and belowthe lower end of the capillary (as shown in FIG. 25)

Total volume=π*((R ²)*L ₃(L ₁)*(3*(R)²+(L ₁)²)/6+(L ₄ −L ₃)*(3*(R)²+(L ₄−L ₃)²)/6)

Example 9: Bubbles

Bubbles in liquid samples or reagents cause variable reductions involume of liquid metered. In small capillaries bubbles when smaller thanthe capillary cross section, are spherical. When they are bigger theyoccupy a cylindrical space (in a cylindrical capillary) and havehemispherical ends.

Case 1: Bubble is not big enough to span the width of the capillary (asshown in FIG. 26)

Subtract bubble volume=(4/3)*π*r ³

Case 2: Bubble occludes the entire width of the capillary (as shown inFIG. 27)

Subtract bubble volume=4π*R ³ +π*R ² *L

Example 10: Blood Outside the Capillary Tip

Case 1: Pendant blood or reagents outside a vertical capillary can causemajor problems in assays since it represents an out-of-controlsituation. As shown in FIG. 28, imaging can easily recognize thissituation.

Case 2: Blood outside the capillary other than pendant

Residual blood outside the capillary also is problematic since it is apotential source of contamination of reagents and of extra volume. Againimaging can recognize the situation.

Example 11: Residual Blood Inside the Capillary Once Sample Dispensinghas Occurred

This can be dealt with by estimating the residual volume and subtractingfrom the total sample volume. FIG. 29 shows an example of a capillarywith residual blood.

Residual volume=π*R ² *L

Example 12: Evaluation of Red Cell Separation from Blood Samples

In many assays it is desirable to remove red cells from the sample thusmaking plasma. When this is done, it is desirable, especially in POCdevices, to know that the separation was effective and to determine thatthere is sufficient plasma to perform the assay.

FIG. 30-FIG. 39 show a schematic of one preferred embodiment of red cellremoval suited to POC devices of the present invention. Magnetizableparticles have antibody to red cells mixed with free antibody to redcells are provided as a dried preparation in the well that will receivea blood sample, as shown in FIG. 30. When a blood sample is added to thewell (shown in FIG. 31) and mixed with the magnetic reagent (shown inFIG. 32, FIG. 33, and FIG. 34), the red cells agglutinate with themagnetic particles and can be removed by placing the blood-containingwell in proximity to a strong magnet (shown in FIG. 35). Byappropriately moving the well relative to the magnet, the red cells areseparated from plasma (shown in FIG. 36) which can then be ejected intoa receiving well for use in an assay (shown in FIG. 37, FIG. 38, andFIG. 39). It is evident that the imaging analysis can determine howeffectively the separation was effected and estimate the volume ofplasma available for assay.

Example 13: Images of Liquid Samples in Capillaries

FIG. 40 shows a high contrast image of a cylindrical tip containing aliquid with low absorbance.

FIG. 41 shows an image of a conical tip containing a liquid with highabsorbance.

FIG. 42 shows a tip with a high absorbance liquid showing two menisciwithin the tip.

FIG. 43 shows a tip with a sample liquid and large bubbles that span thediameter of the tip.

FIG. 44 shows a tip containing water showing a clear upper meniscus in atransparent tip or capillary.

FIG. 41 was analyzed for the length of the liquid column (correspondingto 5 uL) and the resolution of the upper liquid meniscus (Definition ofthe position of the meniscus with >90% confidence). The precision of themeniscus location corresponded to <1% of the length of the liquidcolumn.

Dimensions Pixel widths Length 276 Resolution of meniscus 2 Precision0.7%

Example 14: Effect of Insufficient Sample Volume on Assay Result

The system was used to measure Protein-C in blood. The sample volumeinserted into the system was designed to be 20 uL when the sampletransfer device was used properly. The instrument was set up to use 10uL of blood from this sample. The analyte concentration calculated bythe system is shown in FIG. 45 as the sample volume was deliberatelydecreased from the target level. The result was essentially constantuntil the sample volume was less than the volume required.

Example 15: Sample Transfer Device

FIG. 46 shows an example of a sample transfer device. The deviceconsists of (a) a capillary (made of glass or plastic) optionally coatedwith an anticoagulant or other reagent suitable for pre-analyticaltreatment of samples, (b) a housing which holds the capillary fittedwith (c) a plunger (piston) which can slide within the housing and has araised feature which slides within a groove in the housing, (d) a groovein the housing which engages the piston feature and limits the axialmotion of the plunger so that its motion stops once the sample has beendisplaced and (e) a vent in the housing normally open which is blockedwhen the plunger is activated (moved towards the distal end of thedevice) so as to displace any liquid in the capillary.

FIG. 47 shows a sample transfer device with its capillary filled withsample. The “fill to” location is indicated.

FIG. 48 shows a sample transfer device with sample displaced by movementof the plunger.

FIG. 49 shows a sample transfer device after a sample has beenincompletely ejected.

Example 16: Measurement of Volume by Image Analysis

Known volumes of a liquid sample were aspirated into sample tips using apipetting device. Images of the tips were collected using a commercialflatbed scanner (Dell) and the distances (a) from the distal end of thetip to the meniscus and (b) from the distal end of the tip to thefeature marked on the image below measured. The orientation of the tipand its position relative to the scanner platen were not controlledsince the image was analyzed by measuring the ratio of distance (a) todistance (b) as a measure of sample volume. Locations of the tip,meniscus and feature were measured using commercially imaging software(Jasc). The image was oriented horizontally using the software beforethe locations were recorded and could be read directly on a scaleprovided by the software. FIG. 50 shows an exemplary image.

Distance L1 was the location of the tip

Distance L2 was the location of the meniscus

Distance L3 was the location of the arrow indicated in FIG. 50.

Calc. Vol. Distances in arb units Vol. uL L1 L2 L3 ΔL2-1 ΔL3-1 Ratio uL10.0 120 374 590 254.00 470 0.540426 9.7 12.5 112 400 584 288.00 4720.610169 12.9 15.0 156 470 636 314.00 480 0.654167 15.2 17.5 171 505 654334.00 483 0.691511 17.3 20.0 114 469 596 355.00 482 0.736515 20.0 25.0214 600 694 386.00 480 0.804167 24.5 30.0 165 585 640 420.00 4750.884211 30.3

As shown in FIG. 51 the volume was simply related to the ratio ofdistances and could be calculated. The volume estimate was within lessthan 2% of the actual volume on average.

Example 17: Images of Blood Centrifuged in a Tip

Hematocrits were determined from digital images by measuring the ratioof length of the column of packed red cells and the total liquid column(tip to meniscus). This is easily achieved by feature recognitionsoftware which orients the tip to a known direction and counts pixelsbetween the features. For the tips above, the distances corresponded toseveral hundred pixels permitting a precise measurement.

FIG. 10 shows an empty capped sample tip.

FIG. 11 shows a capped sample tip containing a sample of blood.

FIG. 12 shows a capped sample tip containing a sample of 23% hematocritblood after centrifugation.

FIG. 13 shows a capped sample tip containing a sample of 31% hematocritblood after centrifugation.

FIG. 14 shows a capped sample tip containing a sample of 40% hematocritblood after centrifugation.

FIG. 15 shows a capped sample tip containing a sample of 52% hematocritblood after centrifugation.

FIG. 16 shows a capped sample tip containing a sample of 68% hematocritblood after centrifugation.

FIG. 17 shows a comparison of hematocrit measured using by the digitallyimaging system a centrifuged sample (hematocrit, % reported) andhematocrit measured by standard techniques (hematocrit, % target). Anexample of a standard technique of hematocrit measurement may includemicrohematocrit measurement in a glass capillary using a standardlaboratory centrifuge and measuring the length of the packed red cellbed and the total length of capillary occupied by the sample.

Example 18: System Including Components for Blood Separation

A system designed for separation of blood can include the followingfeatures:

1. Design of the tip shape.

-   -   a. Aspect ratio about 20:1 length:diameter provides convenient        lengths for measurement of sample, packed cell and plasma        volumes    -   b. Shaped tips enable tight sealing with a vinyl cap and easy        removal of cap if needed.    -   c. Slight draft angle conical shape of main part of tip and        wider conical upper section of tip are optimal for insertion of        the plasma recovery means. Note that the “counter radial” design        (tip is narrower at the end distal to the axis of rotation) is        unusual.    -   d. Wider conical upper section of tip is configured to be        accommodated on an automated pipetting and x-y-z movement stage.        Connection to form a fluid tight connection and easy removal        when needed are facilitated.

2. Use of a very precise and accurate x-y-z stage to move the plasmarecovery tip.

3. Use of imaging technology automatically to control the operations ofcentrifugation and plasma recovery. Movement of the plasma recovery tipto within less than a millimeter of the packed cell-plasma interface.

Example 19: Use of Image Measurement of Liquid Volumes to Improve AssayCalibration

Automatic pipetting devices are generally accurate and precise to about5% or better in the range (say) 5-50 uL. In many assays, volume accuracyand precision have both to be very good (say <2%) to obtain the requiredaccuracy and precision of analyte measurement. Metering and delivery of(1) liquids at volumes less than 5 uL (highly desirable when maximum useof a small volume sample is required), and (2) liquids having“problematic” physical properties (such as viscous solutions, solutionscontaining detergents etc.) can often have poor precision and accuracywhich compromises the accuracy of assay results. One inventive solutionto these issues is to use image analysis of the liquids to measure thevolume of liquids (samples, diluents, reagents, calibrators andcontrols) and then to correct the assay calibration function to allowfor deviations (up to [say] 20%) from the intended volume. We have shownthat in pipette tips which have very precise dimensions, volumes assmall as 5 uL can be measured with very good accuracy and precision(<2%). Below we document (1) volume measurement accuracy and precisionand (2) use of known relationships between the volumes of solutions usedin assays (samples, reagents etc.) and assay response to correct thecalibration of assays.

(1) Accuracy of Volume Measurement by Image Analysis:

In the table below, known volumes of a solution of bromphenol blue wereaspirated into conical tips.

The solutions were positioned in the middle portion of the tip andimaged using a scanner. Tip orientation and position were determined bystandard methods. The tip orientation was adjusted by an algorithm andthe length of the liquid column measured. Using the known internaldimensions of the tip, the liquid volume was calculated. Four replicateimages were taken for four aliquots in four tips. The error given belowtherefore reflects image reproducibility and tip dimensional accuracyand precision.

Target volume Measured volume Total Error uL uL % 5 5.01 1.39 20 19.981.80

Note that the volume measurement does not rely on accurately positioningthe liquid in the tip. Image analysis provides information as to thelocation of the liquid in the tip. Knowing the dimensions of the tip onecan always compute the volume from the portion of the tip occupied bythe liquid wherever it is.

(2) Correction of Assay Calibration by Incorporation of Liquid VolumeMeasurement

This is achieved as illustrated in the following simulation. Consider anassay in which a sample is combined with two reagents (called 1 and 2).The target volume for sample, reagent 1 and reagent 2 is 10 uL. Theassay result is calculated from a standard curve relating measuredsignal to analyte concentration. As part of the assay calibrationprocess the experiments can be performed in which volumes used forsample and reagents are changed to 8 and 12 uL in addition to 10 uL andassay results calculated based on the calibration appropriate for 10 uLvolumes. In FIG. 105, FIG. 106, and FIG. 107, the results were plottedagainst actual volumes. For the sample volume, the assay result isessentially directly proportional to the volume used (shown in FIG.105). For the reagents, somewhat non-rectilinear responses were seen(shown in FIG. 106 and FIG. 107). These responses are based on “typical”assays well-known in the field and the magnitude of the changes withvolume are representative.

We then simulate an evaluation in which we have imposed a degree ofrandom error (about 5%, to reflect a “real world situation”) on assayresults in addition to the effects of the use of inappropriate volumes.We include results in which sample, reagent 1 and reagent 2 volumes areset at 8, 10 and 12 uL in all combinations. When the results from thisexercise are plotted as shown in FIG. 108 without correction for volumeerrors, as would be expected there is a significant error in thereported result due to ignoring the fact that the actual volumes usedwere different from those used for assay calibration.

When we allow for the volume variances from target values using theknown assay response to volumes, and plot corrected analyte values weobtain the much improved result shown in FIG. 109, however. This wasachieved by multiple regression analysis of the data.

Summary statistics comparing results calculated with and without volumecorrection are presented in the table below and reflect a significantimprovement in all metrics by the use of volume correction. SEE is thestandard error of the estimate.

Correlation Analyte Regression coefficient CV SEE/Mean Volume correctionslope R{circumflex over ( )}2 % % No correction 0.994 0.914 13.3 16.5Correction 0.999 0.994 3.6 7.4

Example 20: Hemagglutination Inhibition Assay Read Using Microscopy andImage Analysis

In phosphate buffered saline (100 uL), containing 0.3% w/vglutaraldehyde stabilized turkey red blood cells, and 0.5 mg/mL bovineserum albumin and, where indicated, 2 hemagglutination units ofinactivated influenza virus and 15 ug goat polyclonal anti-influenza Bantibody were incubated for 15 minutes in a conical bottom PCR tube atRT. The reaction product from the bottom of the tube was transferred toa transparent slide illuminated with white light and imaged with a4-fold magnification using a digital camera. As may be seen in FIG. 131,agglutination caused by reaction of the red cells with thehemeagglutinin of the virus (FIG. 131 sample 3) is easily observable bycomparison with an unagglutinated control (FIG. 131 sample 1). Whenexcess antibody to the virus was also present agglutination wascompletely inhibited. Two effects of the agglutination reaction arenotable: (1) in agglutinated samples, there are more red cells due tothe more rapid sedimentation of the agglutinates compared with thecontrol and (2) each red cell is on average more clustered with otherred cells. The agglutination reaction can be quantified following imagerecognition software to identify, locate and count red cells andagglutinates.

FIG. 131 sample 1 shows a non-agglutinated control (no virus, noantibody). FIG. 131 sample 2 shows non agglutinated sample (virus plusantibody). FIG. 131 sample 3 shows an agglutinated (virus, no antibody).

Example 21: Sample Preparation, Examination of Supernatant Quality andEstimation of the Quality of the LDL Precipitate

Plasma was diluted (1:10) into a mixture of dextran sulfate (25 mg/dL)and magnesium sulfate (100 mM) then incubated for 1 minute toprecipitate LDL-cholesterol. The reaction product was aspirated into thetube of a centrifuge, capped then and spun at 3000 rpm for threeminutes. Images were taken of the original reaction mixture prior tocentrifugation (showing the white precipitate), following centrifugation(showing a clear supernatant) and of the LDL-cholesterol pellet (afterremoval of the cap). For example, FIGS. 132, 134 illustrate examples ofimages taken of reaction product.

Images of the LDL-precipitation reaction product were analyzed asfollows. The pixel color levels were plotted as a function of theirvertical position. The variance of the values was measured and valuesfor the three colors summed. Because of the particles of precipitatedLDL, which strongly scatter light, the precipitate value (1154) was muchgreater than (672) of the clear supernatant. Comparison of thesupernatant value with that of a control no exposed to the precipitationreagent allows the quality of the centrifugation to be evaluated (datanot shown). FIG. 133 provides examples of images that were analyzedbefore spinning in the centrifuge, and after spinning in the centrifuge.

After removal of the black vinyl cap, an image of the LDL precipitatewas taken. Its volume can be measured quite accurately, knowing thegeometry of the tip and the size of a pixel in the image. In thisexperiment, the volume of the precipitate was estimated as 0.155+/−0.015uL.

Example 22: Improving the Performance of an Assay for AlanineAminotransferase (ALT) by Use of 3-Color Image Blanking of OpticalSignals Due to the Sample

ALT in serum can be measured in an assay in which the enzyme convertsalanine to pyruvate which is in turn used to make hydrogen peroxide withoxygen and pyruvate oxidase. The peroxide is then used to make a coloredproduct by the enzyme horseradish peroxidase, aminoantipyrene andN-Ethyl-N-(3sulfopropyl)aniline. The colored product absorbs maximallyat 560 nm.

It was found that some serum samples have significant absorbance at thiswavelength as shown in FIG. 135 and accordingly interfered with theassay. In particular when using relatively high sample concentrations(such as a final dilution in the assay of 1:10), 3-color image analysisof the ALT assay gave poor results with clinical samples.

FIG. 135 illustrates spectra of several serum samples diluted 1:10 intobuffer; OD is plotted against wavelength (nm). Great variation of OD isillustrated in FIG. 135.

In conventional spectroscopy, this issue is dealt with by taking a blankreading of the sample without the assay ingredients added andsubtracting the blank from the signal generated by the assay. In 3-colorimage analysis as in the present invention, it has been found that ananalogous method can be used. The diluted sample may be imaged andthree-color values extracted. The assay calibration algorithm may thenbe changed to include the signals from the unreacted sample.Specifically in this case, the original algorithm (not including sampleblank signal) was ALT concentration=a+b*R+c*G+d*B+e*R² where a, b, c, dand e are empirically derived constants and R, G, B are the signalvalues in red, green and blue channels respectively. The improvedalgorithm was: ALT concentration=a+b*R+c*G+d*B+e*Rs, +f*Rs² where Rs isthe signal from the sample blank in the red channel (note that theempirically derived values of a, b, c, d and e were different from thoseof the original algorithm).

When 21 serum samples ranging in ALT activity from 0 to 250 U/L weremeasured in triplicate and results of 3-color image analysis comparedwith those provided by a clinical laboratory method (Teco) the followingregression statistics were obtained indicating much-improved results.

Calibration method R{circumflex over ( )}2 Slope Intercept (U/L) SEEOriginal 0.922 0.922 4.5 18.9 Improved 0.972 0.972 1.6 10.0

Example 23: Speeding-Up and Providing Objective Analysis of aHemagglutination Inhibition Assay for Anti-Influenza Antibodies

Reaction mixtures prepared as described in Example 20, were incubatedfor only one minute then introduced into three separate micro channels(as described for the cytometry examples above) and imaged. About 5-10images are taken for each sample, in order to get adequate statistics.On average, each image consists of around 800-900 cells. Theagglutination process can be objectively evaluated by measuring theradial distribution of cells around a representative selection ofindividual cells using the function.

The images were processed to obtain centroid positions of the individualcells in 2D space. The 2D positions were used to compute a radialdistribution function (RDF), also known as a pair correlation function.The radial distribution function, g(r) quantifies the probability offinding a cell at a distance r from the selected cell. Mathematically,

g(r)=ρ(r)/ρ_(o)

where ρ(r)2πrdr is the number of cells found at a distance of r from aparticular cell and ρ_(o) is the average cell density over the entireimage window. The value g(r) is calculated as an average over allparticles in the image and over multiple images, to ensure astatistically meaningful result.

Results

The value of the first peak of g(r) quantifies the number of doublets inthe sample. Hence, the g(r) for the agglutinated sample should be higherin magnitude compared to the other two samples. The value of g risesquickly from 0 to a maximum over a distance of about 20 pixels(corresponding to about 12 um about twice the diameter of a red cell)then declines to about 1.0. As shown below, the agglutination due tovirus was distinguishable from no agglutination when virus is absent orantibody inhibits the virus-induced agglutination.

Virus Antibody Agglutination gmax None None No 1.44 Present None Yes1.67 Present Present No 1.47

Example 24: Preparation of Analyte Detection Systems Using Aptamers

Two oligo DNA aptamers were designed to selectively capture proteins(thrombin and insulin). The oligo DNA aptamer was composed of a bindingsite having a sequence selected from published data, an inert portion toextend the binding site from the surface of the bead or microarray, anda reactive group to chemically immobilize the aptamer to the surface.Aptamer 1 (specific for thrombin) had the following sequence:5′-Am-(T)₄₅GGTTGGTGTGGTTGG-3′. Aptamer 2 (specific for insulin) had thefollowing sequence: 5′-Am-(T)₃₂ACAGGGGTGTGGGGACAGGGGTGTGGGG-3′. The “Am”at the beginning of each sequence represents an amino group.

The two aptamers were immobilized on polystyrene beads (5 um)functionalized with carboxyl groups. The beads were then washed and theexcess reagent removed. The beads were then mixed with oligo DNA probesfluorescently labeled and complementary to the binding sites of theaptamers. Hybridization of the probes with the aptamers was detected byfluorescence emission. Only the complementary probe showed a positivehybridization event, as measured by mean fluorescence emission.Hybridization specificity is illustrated by comparison of FIG. 139,which shows beads after hybridization with complementary probe, to FIG.140, which shows beads after hybridization with non-complementary probe.Detection was performed with a laser excitation at 635 nm, and emissionfiltered at 650 nm (±10 nm) on a CCD camera, after deposition of thebeads on the analysis substrate. A similar procedure was used on a glasssurface coated with epoxysilane to immobilize Aptamers 1 and 2. Thearray was hybridzed with the fluorescent probe and the specificrecognition of the aptamer binding site measured by fluorescenceemission detection with a CCD camera set-up and an Array Scanner(Inopsys). FIG. 141 illustrates the binding specificity of the aptamerson the array, with more detail illustrated in FIG. 142. FIG. 143 showsan example array scan.

Example 25: Detection of Analyte Using Aptamers

An array comprising Aptamer 1 hybridized with fluorescently labeled,complementary probe was prepared as in Example 24. Thrombin wasintroduced to the array at a concentration of about 100 nM and allowedto react with the Aptamer 1-probe complex. The fluorescent emissionsignal from Aptamer 1 on the array was reduced by 2.5 fold, indicatingdisplacement of the probe by binding of Aptamer 1 and thrombin.

Example 26: Binders

Two types of binder are biotinylated and used to create capture surfaceson an assay unit solid-phase coated with avidin. Assay reagentproduction and luminescence-readout assay results are obtained using (1)aptamers and (2) single-chain Fv antibody fragments (SCFVs) onmicrotiter plates. Aptamer and SCFVs as binders for luminescence-basedassays are adaptable to tips and imaging systems and devices providedherein. Analytes can be assayed and read using cameras to measure colorby changing the signal-generating reagent from alkaline phosphatase to,e.g., horse-radish peroxidase with a chromogenic substrate or usingalkaline phosphatase with a chromogenic substrate. Tips in microtiterplate (or other formats) can be read in any cartridge (assay unit)format.

Example 27: Vitamin D Assay Using DNA Aptamers on Microtiter Plate

In this example, an assay for vitamin D is performed usingsingle-stranded DNA aptamers. Biotinylated DNA aptamers are coated on aultravidin coated polystyrene surface of a microtiter plate having aplurality of wells. Before coating, the aptamers are quickly denaturedand renatured by heating at about 95 degrees Celsius, then immediatelycooled on ice. About 15 microliters of the refolded biotinylated vitaminD DNA aptamers in 25 mM Tris, containing NaCl, MgCl2, 10% Ethanol, pH7.5, are then added into each well to form the capture surface. Aftercoating, the wells are washed and blocked with about 100 uL of ablocking reagent to reduce nonspecific binding.

The analyte for the assay (vitamin D) is diluted in Tris, NaCl, MgCl2,10% Ethanol, pH 7.5, and is mixed with a solution of vitamin D-AlkalinePhosphatase conjugate at different concentrations in the desired assayrange, and provided to the assay unit for 10 minutes incubation at roomtemperature. The assay unit is then washed three times with 100 uL ofwash buffer. About 40 uL of substrate for Alkaline phosphatase is addedto each assay well and chemiluminescence data (table below) is collectedafter about 10 minutes. FIG. 144 is a plot of chemiluminescence againstthe concentration (ng/ml) of vitamin D.

Vitamin D (ng/ml) 0 1 100 200 Chemluminescence 155674.1 113824.349346.13 33824.27 (RLU) 159471 110794.2 49699.04 35794.18 162650.3101655.7 53158.25 36655.66 159920.8 99266.41 50195.63 35166.41 Avg159429.1 106385.1 50599.76 35360.13 cv % 1.80 6.60 3.44 3.37 B/B0 100%67% 32% 22%

Example 28: Estradiol Assay on Microtiter Plate

In this example, an assay is performed for a steroid hormone (estradiol)using single-chain variable fragments (scfv). In this assay, the innersurface of the assay unit is coated with biotinylated scFv on ultravidincoated polystyrene surface of a microtiter plate having a plurality ofwells. About 15 microliters of 1 ug/ml biotinylated scFv in Trisbuffered Saline, pH 8, 0.03% BSA, 0.05% Thimerasol were added to eachassay unit. After washing, each assay unit is fixed with 100 uL Fixativereagent followed by an overnight dry with dry air and stored dessicated.

The analyte for the assay (free estradiol) is diluted in Tris bufferedSaline, pH 8, BSA, Thimerasol, mixed with an estradiol-AlkalinePhosphatase conjugate, in stabilizer from Biostab, and is provided tothe assay unit coated with the scfv for about 10 minutes at roomtemperature.

The assay wells are then washed 5 times with 100 uL of wash buffer.After the washes, 40 uL of luminogenic substrate for Alkalinephosphatase (KPL PhosphaGlo) is added to each assay unit andchemiluminescence data (table below) is collected after about 10minutes. FIG. 145 is a plot of chemiluminescence against theconcentration (pg/ml) of estradiol.

Estradiol (pg/ml) 0 20 200 2000 Chemluminescence 5505.454 1997.885493.864 389.863 (RLU) 5505.454 2005.112 496.932 374.317 5659.6131739.771 503.25 417.021 avg 5557 1914 498 394 % cv 1.6 7.9 1.0 5.5 b/bo100% 34% 9% 7%

Example 29: White Blood Cell Count and Differential Assay

The concentration of white blood cells (WBCs) in the peripheral blood ofhuman subjects can range from about 1000 cells/ul to 100,000 cells/ul.However, in some cases the range of the imaging system is more limited,such as from about 4000 cells/ul to 7000 cells/ul. If the cellconcentration is less than 4000 cells/ul, the system may not be able toenumerate a target of 10,000 cells, as may be required by the assay. Ifthe cell concentration in the sample is more than 7000 cells/ul, eachfield of view may be too crowded to perform accurate image segmentationand cell enumeration. An exemplary approach for imaging WBCs is providedbelow.

In an example, an imaging system (e.g., cytometer) is providedconfigured for fluorescence spectrophotometry. The system usesfluorescence spectrophotometry to measure the cell concentration in thesample. The sample is labeled with fluorescently conjugated antibodiesfor imaging (e.g., AF647-CD45) and also with a fluorescent nucleic acidmarker (e.g., DRAQ5). A quantitative fluorescence readout on thespectrophotometer module provides a measurement of the concentration ofWBCs at low sensitivity (LLOQ of about 5000 cells/ul) but high dynamicrange (e.g., 5000-100,000 cells/ul). A concentration measured on thespectrophotometer allows the calculation of the optimal dilution ratiosuch that the final concentration of the cell suspension is between4000-7000 cells/ul.

FIG. 146 shows the high dynamic range of fluorescence in thespectrophotometric measurement of WBC concentration. WBCs tagged withfluorescently labeled anti-CD45 and other antibodies were excited withred light having a wavelength of about 640 nm and the quantitativefluorescence emission spectrum was collected. Integrated fluorescence isplotted on the y-axis.

Example 30: Streptococcus Group A Detection by Isothermal Amplification

Isothermal amplification of specific genomic samples can be detected byturbidity. In this example, a genomic sample extracted fromStreptococcus group A (StrepA) cells (stock concentration=2×10⁸ org/mlfrom My biosource) was amplified by isothermal amplification and theprogress of the reaction measured by Turbidity. About 5 ul of stockbacterial cells and 45 ul RT PCR grade Water (10× dilution of stock)were heat treated at about 95 degrees Celsius from about 8 to 10 minutes(Cell ruptures and releases the DNA). The genomic sample was diluted andintroduced in a sample volume of about 25 ul in a PCR tube containingreagents for amplification (e.g., DNA Polymerase, Primers, Buffer). ThePCR tube was incubated at about 61 degrees Celsius for about 60 minuteswhile the progress of the reaction was recorded by turbidity. Theresults are as follows, and FIG. 147 shows plots of turbidity as afunction of time:

Conc. (copies/uL) T(min) St. dev (min) 800 24.0 1.6 80 28.3 2.9 0 n/an/a

Three separate experiments were conducted at 800 copies/uL and 80copies/uL. Experiment A was performed using StrepA having a syntheticgenomic DNA template (from Genescript). Experiment B was performed bydiluting stock StrepA 10-fold followed by heat inactivation at 95degrees Celsius from about 8 to 10 min, and followed by serial dilutionof heat inactivated ten-fold diluted stock StrepA. Experiment C wasperformed using a variable concentration of stock StrepA (inactivatedbacterial cells) followed by heat inactivation at 95 degrees Celsius forabout 10 min. The inflections points for each experiment are shown inFIG. 148. For each of 800 copies/uL and 80 copies/uL, a grouping ofthree plots includes Experiment A at the left, Experiment B in themiddle and Experiment C at the right. The average inflections points areprovided in the following table:

Experiment A Experiment B Experiment C AVG STDEV AVG STDEV AVG STDEV 800cp/uL 23.1 0.6 24 1.6 21.1 0.4  80 cp/uL 27 1.4 28.3 2.9 27.2 1.8

Example 31: Use of Magnetic Beads

In this example, magnetic beads are used for the analysis of proteinsand small molecules via ELISA assays. FIG. 110 schematically illustratesan exemplary method for the ELISA assay. The assays include twoproteins, Protein 1 and Protein 2. Protein 1 has a sample dilution ofabout 150-fold (tip protocol=30-fold), a sample volume of about 0.007uL, a diluted sample volume of about 1 uL, a reaction volume of about 3uL, and a reaction time of about 10 minutes (min) (sample incubation andsubstrate incubation). Results for Protein 1 are shown in the followingtable. The results of Test 2 for Protein 1 are shown in FIG. 149.

Conc. % (ng/mL) Test 1 Test 2 Test 3 Cal 1 Cal 2 Cal 3 Recov 1 Recov 2Recov 3 CV 40 59316 46862 57396 40 40 40 99 99 99 0.3 20 25120 2522525099 21 20 20 104 102 102 1.2 10 10551 11360 11463 9 10 10 92 96 97 2.65 5940 5607 5825 5 5 5 106 102 102 2.2 2.5 2476 2588 2497 2.5 2.5 2.5 9999 100 0.3 0 190 166 166

Protein 2 has a sample dilution of about 667-fold, a sample volume ofabout 0.0015 uL, a diluted sample volume of about 1 uL, a reactionvolume of about 3 uL, and a reaction time of about 10 min (sampleincubation and substrate incubation). Results for Protein 2 are shown inthe following table. The results of Test 1 for Protein 2 are shown inFIG. 150.

Conc % (ng/ml) Test 1 Test 2 Cal 1 Cal 2 Recov 1 Recov 2 CV 200000322161 381202 203030 172490 102 86 12 100000 232455 310876 107910 117056108 117 6 50000 133290 192460 43286 52415 87 105 13 25000 89282 10164324919 21908 100 88 9 12500 49856 59574 12576 12041 101 96 3 4000 1592618350 4117 4059 103 101 1 1000 4547 4722 1140 1124 114 112 1 200 12381229 163 172 82 86 4 20 504 458 22 21 109 106 2 0 302 292

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications can be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense.

Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. Various modifications in form and detail of theembodiments of the invention will be apparent to a person skilled in theart. It is therefore contemplated that the invention shall also coverany such modifications, variations and equivalents.

1.-91. (canceled)
 92. A centrifugation system, comprising: a pipette tipcomprising: a circular top opening; a first segment having a taperedsidewall and extending from the top opening; and a second segmentextending from a bottom of the first segment, the second segment beingnarrower than a narrow most portion of the first segment and having atleast one aperture disposed at a bottom surface; and a centrifugationvessel comprising: a first segment having a tapered sidewall configuredto mate with the first segment of the pipette tip; and a second segmentextending below the first segment and being narrower than the firstsegment, the second segment being below a topmost portion of the secondsegment of the pipette tip when fully mated with the pipette tip. 93.The centrifugation system of claim 92, wherein the centrifugation vesselincludes a bottom surface configured to support the bottom surface ofthe pipette tip.
 94. The centrifugation system of claim 93, wherein thebottom surface of the centrifugation vessel includes at least onesupport element protruding from the bottom surface and configured tocontact the bottom surface of the pipette tip.
 95. The centrifugationsystem of claim 94, wherein the at least one support element includes asubstantially semispherical nub.
 96. The centrifugation system of claim95, wherein the at least one aperture of the pipette tip is centrallylocated on the bottom surface of the pipette tip, and wherein at leastone support element includes a plurality of substantially semisphericalnumbs configured to contact an edge of the bottom surface of the pipettetip without occluding the aperture.
 97. The centrifugation system ofclaim 94, wherein the at least one aperture of the pipette tip iscentrally located on the bottom surface of the pipette tip, and whereinat least one support element includes a support ring extending around aninner circumference of the bottom surface of the centrifugation vesselconfigured to contact the bottom surface of the pipette tip around acircumference of the aperture.
 98. The centrifugation system of claim92, wherein the at least one aperture of the pipette tip includes aplurality of apertures.
 99. A pipette tip for mating with acentrifugation vessel, comprising: a circular top opening; a firstsegment having a tapered sidewall and extending from the top opening;and a second segment extending from a bottom of the first segment, thesecond segment being narrower than a narrow most portion of the firstsegment and having at least one aperture disposed at a bottom surface.100. The pipette tip of claim 99, wherein the at least one apertureincludes a single central aperture disposed in a center of the bottomsurface.
 101. The pipette tip of claim 100, further comprising at leastone contact surface located on the bottom surface and configured tocontact at least one support element of the centrifugation vessel. 102.The pipette tip of claim 99, wherein the at least one aperture includesa plurality of apertures disposed around the bottom surface.
 103. Thepipette tip of claim 102, further comprising a plurality of contactsurfaces disposed between the plurality of apertures configured tocontact at least one support element of the centrifugation vessel. 104.The pipette tip of claim 99, wherein the second segment is longer than adistance between a contact surface of the centrifugation vessel and amax fluid height of the centrifugation vessel.
 105. The pipette tip ofclaim 101 wherein the at least one contact surface is tapered relativeto the bottom surface.
 106. A centrifugation vessel for mating with apipette tip, comprising: a first segment having a tapered sidewall; anda second segment extending below the first segment and being narrowerthan the first segment, the second segment extending higher than aninjection segment of the pipette tip.
 107. The centrifugation vessel ofclaim 106, further comprising a bottom surface configured to support thepipette tip.
 108. The centrifugation vessel of claim 107, wherein thebottom surface includes at least one support element attached theretofor contacting a bottom surface of the pipette tip.
 109. Thecentrifugation vessel of claim 108, wherein the at least one supportelement includes support ring configured to extend around acircumference of an aperture of the pipette tip.
 110. The centrifugationvessel of claim 108, wherein the at least one support element includes aplurality of support nubs, each nub being substantially semisphericaland configured to engage a tapered contact surface of the pipette tip.111. The centrifugation vessel of claim 108, wherein the at least onesupport element includes a central support nub having a substantiallysemispherical shape and being configured to engage the bottom surface ofthe pipette tip.